Bispecific antibodies specific for pd1 and tim3

ABSTRACT

The invention relates to bispecific antibodies comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, in particular to bispecific antibodies, wherein the bispecific antibody binds to 5 TIM3 with a lower binding affinity when compared to the binding to PD1. The invention further relates to methods of producing these molecules and to methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/367,017, filed Mar. 27, 2019, pending, which is a divisional of U.S.application Ser. No. 15/280,372, filed Sep. 29, 2016, now U.S. Pat. No.10,287,352, which claims the benefit of priority to European PatentApplication No. 15188036.6, filed Oct. 2, 2015, and European PatentApplication No. 15188065.5, filed Oct. 2, 2015, each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing with 94 sequencesand submitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Aug. 11, 2021, is namedP33115-US-2_SeqList.txt, and is 147,703 bytes in size.

FIELD OF THE INVENTION

The invention relates to bispecific antibodies comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, in particular tobispecific antibodies, wherein the bispecific antibody binds to TIM3with a lower binding affinity when compared to the binding to PD1. Theinvention further relates to methods of producing these molecules and tomethods of using the same.

BACKGROUND

The importance of the immune system in protection against cancer isbased on its capacity to detect and destroy abnormal cells. However,some tumor cells are able to escape the immune system by engendering astate of immunosuppression (Zitvogel et al., Nature Reviews Immunology 6(2006), 715-727). One example of a mechanism of immunosuppressionpresent in tumor-bearing hosts is the promotion of T cell dysfunction orexhaustion. T cells have been the major focus of efforts totherapeutically manipulate endogenous antitumour immunity owing to theircapacity for the selective recognition of peptides derived from proteinsin all cellular compartments; their capacity to directly recognize andkill antigen-expressing cells (by CD8+ effector T cells; also known ascytotoxic T lymphocytes (CTLs)) and their ability to orchestrate diverseimmune responses (by CD4+ helper T cells), which integrates adaptive andinnate effector mechanisms. Exhausted T cells fail to proliferate andexert effector functions such as cytotoxicity and cytokine secretion inresponse to antigen stimulation. Further studies identified thatexhausted T cells are characterized by sustained expression of theinhibitory molecule PD-1 (programmed cell death protein 1) and thatblockade of PD-1 and PD-L1 (PD-1 ligand) interactions can reverse T cellexhaustion and restore antigen-specific T cell responses inLCMV-infected mice (Barber et al., Nature 439 (2006), 682-687). However,targeting the PD-1-PD-L1 pathway alone does not always result inreversal of T cell exhaustion (Gehring et al., Gastroenterology 137(2009), 682-690), indicating that other molecules are likely involved inT cell exhaustion (Sakuishi, J. Experimental Med. 207 (2010),2187-2194).

TIM-3 is a molecule originally identified as being selectively expressedon IFN-7-secreting Th1 and Tcl cells (Monney et al., Nature 415 (2002),536-541). The interaction of TIM-3 with its ligand, galectin-9, triggerscell death in TIM-3+ T cells. Thus, both TIM-3 and PD-1 can function asnegative regulators of T cell responses. It has been shown that TIM-3marks the most suppressed or dysfunctional population of CD8+ T cells inpreclinical models of both solid and hematologic malignancy (Sakuishi,J. Experimental Med. 207 (2010), 2187-2194; Zhou, Blood 117 (2011),4501-4510; Majeti R et al., PNAS, 106 (2009), 3396-3401). In thesemodels, all of the CD8+ TIM-3+ T cells coexpress PD1, and thesedual-expressing cells exhibit greater defects in both cell-cycleprogression and effector cytokine production [interleukin (IL)-2, TNF,and IFN-7] than cells that express PD1 alone. Thus, the TIM-3 pathwaymay cooperate with the PD-1 pathway to promote the development of asevere dysfunctional phenotype in CD8+ T cells in cancer. The combinedtargeting of the TIM-3 and PD1 pathways is thus expected to be highlyeffective in controlling tumor growth.

TIM3 is a human protein which belongs to the immunoglobulin superfamily,and TIM family of proteins. In humans, as similar to mice, TIM-3 isexpressed on T-cells as well as phagocytic cells such as macrophages anddendritic cells. Binding of TIM3 to a protein ligand (e.g., galectin-9)can inhibit the Th1 response via mechanism of apoptosis induction, andtherefore lead to such as induction of peripheral tolerance. Thereduction in expression of human TIM3 with siRNA or the inhibition ofhuman TIM3 by blocking-antibody increased the secretion of interferonalpha from CD4 positive T-cells, supporting the inhibitory role of TIM3in human T cells. In phagocytes, TIM3 also functions as a receptor forrecognizing the apoptosis cells. Analysis of clinical samples fromautoimmune disease patients showed no expression of TIM3 in CD4 positivecells. In particular, in T cell clones derived from the cerebrospinalfluid of patients with multiple sclerosis, the expression level of TIM3was lower and the secretion level of IFN-gamma was higher than those ofclones derived from normal healthy persons (Koguchi K et al., J Exp Med.203 (2006), 1413-1418). There are reports on relation of TIM-3 withallergic diseases or asthma (WO 96/27603 and WO2003/063792).

Examples of the anti-TIM3 monoclonal antibodies include anti-human TIM3rat monoclonal antibody (Clone 344823, manufactured by R&D Systems) andanti-human TIM-3 mouse monoclonal antibody (Clone F38-2E2, manufacturedby R&D Systems). WO2013/06490 relates to anti-TIM3 antibodies which showrapid internalization and immunoconjugates thereof for treating cancerand reducing inflammation. US2012/189617 relates to anti-TIM-3antibodies which exhibit higher effector activity such as anantibody-dependent cellular cytotoxicity (ADCC activity) for diseasesrelating to a human TIM3 expressing cell.

Programmed cell death protein 1 (PD-1 or CD279) is an inhibitory memberof the CD28 family of receptors, that also includes CD28, CTLA-4, ICOSand BTLA. PD-1 is a cell surface receptor and is expressed on activatedB cells, T cells, and myeloid cells (Okazaki et al (2002) Curr. Opin.Immunol. 14: 391779-82; Bennett et al. (2003) J Immunol 170:711-8). Thestructure of PD-1 is a monomeric type 1 transmembrane protein,consisting of one immunoglobulin variable-like extracellular domain anda cytoplasmic domain containing an immunoreceptor tyrosine-basedinhibitory motif (ITIM) and an immunoreceptor tyrosine-based switchmotif (ITSM). Activated T cells transiently express PD1, but sustainedhyperexpression of PD1 and its ligand PDL1 promote immune exhaustion,leading to persistence of viral infections, tumor evasion, increasedinfections and mortality. PD1 expression is induced by antigenrecognition via the T-cell receptor and its expression is maintainedprimarily through continuous T-cell receptor signaling. After prolongedantigen exposure, the PD1 locus fails to be remethylated, which promotescontinuous hyperexpression. Blocking the PD1 pathway can restore theexhausted T-cell functionality in cancer and chronic viral infections(Sheridan, Nature Biotechnology 30 (2012), 729-730). Monoclonalantibodies to PD-1 have been described, for example, in WO 2003/042402,WO 2004/004771, WO 2004/056875, WO 2004/072286, WO 2004/087196, WO2006/121168, WO 2006/133396, WO 2007/005874, WO 2008/083174, WO2008/156712, WO 2009/024531, WO 2009/014708, WO 2009/101611, WO2009/114335, WO 2009/154335, WO 2010/027828, WO 2010/027423, WO2010/029434, WO 2010/029435, WO 2010/036959, WO 2010/063011, WO2010/089411, WO 2011/066342, WO 2011/110604, WO 2011/110621, WO2012/145493, WO 2013/014668, WO 2014/179664, and WO 2015/112900.

It has also been shown that blocking both PD1 and TIM3 can restore theantibacterial immune responses, for instance in patients with acutealcoholic hepatitis (AAH). Lymphocytes from these patients express highlevels of immune inhibitory receptors, produce lower levels ofinterferon gamma, and have increased IL10 production due to chronicendotoxin exposure. These effects can be reversed by blocking PD1 andTIM3, which increase the antimicrobial activities of T cells andneutrophils (Markwick et al, Gastroenterology 148 (2015), 590-602).

Bispecific antibodies against TIM3 and PD1 for immunotherapy in chronicimmune conditions have already been described in WO 2011/159877.However, there is a need of providing new bispecific antibodies that notonly simultaneously bind to PD1 and TIM3 and thus selectively target Tcells expressing both PD1 and TIM3, but that also avoid blocking of TIM3on other cells such as innate immune cells, for example naive dendriticcells (DCs) and monocytes. The bispecific antibodies of the presentinvention do not only effectively block PD1 and Tim3 on T cellsoverexpressing both PD1 and TIM3, they are very selective for thesecells and thereby side effects by administering highly active TIM3antibodies may be avoided.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein

-   -   said first antigen-binding site specifically binding to PD1        comprises        -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:37,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:38, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:39; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:40;        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:41, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:42; and    -   said second antigen-binding site specifically binding to TIM3        comprises    -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:1,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:2, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:3; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4            or SEQ ID NO:11 or SEQ ID NO:12,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:5, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:6; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:17,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:18, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:19; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:20,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:21, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:22; or    -   (c) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:29,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:30, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:31; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:32,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:33, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:34.

In one aspect, the bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3 is bivalent.

In another aspect, provided is a bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein thebispecific antibody binds to TIM3 with low affinity and binds to PD1with high affinity. In a particular aspect, the invention provides abispecific antibody comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM3, wherein the bispecific antibody binds toTIM3 with an at least 50 fold lower binding affinity when compared tothe binding to PD1, more particularly with an at least 100 fold lowerbinding affinity when compared to the binding to PD1. In one preferredembodiment the binding affinity (KD) is determined with Surface PlasmonResonance Assay (as described e.g. in Example 12.)

In a further aspect, provided is a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein

-   -   said first antigen-binding site specifically binding to PD1        comprises        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 43 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 44, or        -   (b) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 46, or        -   (c) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 47, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 48, or        -   (e) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 49,    -   and said second antigen-binding site specifically binding to        TIM3 comprises        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 7 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 8, or        -   (b) a VH domain comprising the amino acid sequence of SEQ ID            NO: 9 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 10, or        -   (c) a VH domain comprising the amino acid sequence of SEQ ID            NO: 13 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 14, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 15 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 16, or        -   (e) a VH domain comprising the amino acid sequence of SEQ ID            NO: 23 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 24, or        -   (f) a VH domain comprising the amino acid sequence of SEQ ID            NO: 25 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 26, or        -   (g) a VH domain comprising the amino acid sequence of SEQ ID            NO: 27 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 28, or        -   (h) a VH domain comprising the amino acid sequence of SEQ ID            NO: 35 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 36.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein

-   -   said first antigen-binding site specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 45 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 46,    -   and said second antigen-binding site specifically binding to        TIM3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 15 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 16 or a VH domain comprising the amino acid        sequence of SEQ ID NO: 25 and a VL domain comprising the amino        acid sequence of SEQ ID NO: 26.

Particularly, the invention provides a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein

-   -   said first antigen-binding site specifically binding to PD1        comprises        -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:37,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:38, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:39; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:40;        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:41, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:42; and    -   said second antigen-binding site specifically binding to TIM3        comprises a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:17,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:18, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:19; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:20,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:21, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:22.

More particularly, provided is a bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein said firstantigen-binding site specifically binding to PD1 comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 45 and a VL domaincomprising the amino acid sequence of SEQ ID NO: 46, and said secondantigen-binding site specifically binding to TIM3 comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 25 and a VL domaincomprising the amino acid sequence of SEQ ID NO: 26.

In a particular aspect, the invention provides a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,wherein the bispecific antibody binds to TIM3 with an at least 50 foldlower binding affinity when compared to the binding to PD1, moreparticularly with an at least 100 fold lower binding affinity whencompared to the binding to PD1.

In a further aspect, the bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3 is a human,humanized or chimeric antibody. In particular, it is a humanized orchimeric antibody.

In another aspect, the invention relates to a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,wherein the bispecific antibody comprises an Fc domain, a first Fabfragment comprising the antigen-binding site that specifically binds toPD1 and a second Fab fragment comprising the antigen-binding site thatspecifically binds to TIM3.

In particular, the Fc domain is an IgG domain, more particularly an IgG1Fc domain or an IgG4 Fc domain.

In one aspect, the invention relates to a bispecific antibody comprisinga first antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein the Fcdomain comprises one or more amino acid substitution that reducesbinding to an Fc receptor, in particular towards Fcγ receptor. Inparticular, the Fc domain is of human IgG1 subclass with the amino acidmutations L234A, L235A and P329G (numbering according to Kabat EUindex).

In another aspect, the invention relates to a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,wherein the Fc domain comprises a modification promoting the associationof the first and second subunit of the Fc domain.

In one aspect, the invention relates to a bispecific antibody comprisinga first antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein the firstsubunit of the Fc domain comprises knobs and the second subunit of theFc domain comprises holes according to the knobs into holes method. In aparticular aspect, the first subunit of the Fc domain comprises theamino acid substitutions S354C and T366W (EU numbering) and the secondsubunit of the Fc domain comprises the amino acid substitutions Y349C,T366S and Y407V (numbering according to Kabat EU index).

In an additional aspect, the invention relates to a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,wherein in one of the Fab fragments the variable domains VL and VH arereplaced by each other so that the VH domain is part of the light chainand the VL domain is part of the heavy chain. In a particular aspect,the bispecific antibody is one, wherein in the first Fab fragmentcomprising the antigen-binding site that specifically binds to PD1 thevariable domains VL and VH are replaced by each other.

In a further aspect, the invention is concerned with a bispecificantibody comprising a first antigen-binding site that specifically bindsto PD1 and a second antigen-binding site that specifically binds toTIM3, wherein in one of the Fab fragments in the constant domain CL theamino acid at position 124 is substituted independently by lysine (K),arginine (R) or histidine (H) (numbering according to Kabat EU Index),and in the constant domain CH1 the amino acids at positions 147 and 213are substituted independently by glutamic acid (E) or aspartic acid (D)(numbering according to Kabat EU index). In a particular aspect, thebispecific antibody is one, wherein in the second Fab fragmentcomprising the antigen-binding site that specifically binds to TIM3 theconstant domain CL the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat EU Index), and in the constant domain CH1 the aminoacids at positions 147 and 213 are substituted independently by glutamicacid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In another aspect, the invention provides a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,comprising

-   (a) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 50, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 52, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 51, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:53, or-   (b) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 54, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 56, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 55, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:57, or-   (c) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 58, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 60, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 59, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:61, or-   (d) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 62, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 64, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 63, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:65, or-   (e) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 66, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 68, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 67, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:69.

In a particular aspect, the invention provides a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,comprising

-   (a) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 50, a first light chain comprising the amino acid sequence of    SEQ ID NO: 52, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 51, and a second light chain comprising the    amino acid sequence of SEQ ID NO:53, or-   (b) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 54, a first light chain comprising the amino acid sequence of    SEQ ID NO: 56, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 55, and a second light chain comprising the    amino acid sequence of SEQ ID NO:57, or-   (c) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 58, a first light chain comprising the amino acid sequence of    SEQ ID NO: 60, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 59, and a second light chain comprising the    amino acid sequence of SEQ ID NO:61, or-   (d) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 62, a first light chain comprising the amino acid sequence of    SEQ ID NO: 64, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 63, and a second light chain comprising the    amino acid sequence of SEQ ID NO:65, or-   (e) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 66, a first light chain comprising the amino acid sequence of    SEQ ID NO: 68, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 67, and a second light chain comprising the    amino acid sequence of SEQ ID NO:69.

According to another aspect of the invention, there is provided apolynucleotide encoding the bispecific antibody as described hereinbefore. The invention further provides a vector, particularly anexpression vector, comprising a polynucleotide of the invention and aprokaryotic or eukaryotic host cell comprising the polynucleotide or thevector of the invention. In some embodiments the host cell is aeukaryotic cell, particularly a mammalian cell.

In another aspect, provided is a method for producing a bispecificantibody comprising a first antigen-binding site that specifically bindsto PD1 and a second antigen-binding site that specifically binds to TIM3as described herein, comprising the steps of a) transforming a host cellwith vectors comprising polynucleotides encoding said bispecificantibody, b) culturing the host cell according under conditions suitablefor the expression of the bispecific antibody and c) recovering thebispecific antibody from the culture. The invention also encompasses abispecific antibody produced by the method of the invention.

The invention further provides a pharmaceutical composition comprising abispecific antibody comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM3 as described herein, and at least onepharmaceutically acceptable excipient.

Also encompassed by the invention is the bispecific antibody comprisinga first antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3 as describedherein, or the pharmaceutical composition comprising the bispecificantibody, for use as a medicament.

In another aspect, the invention provides a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3 asdescribed herein, or the pharmaceutical composition comprising thebispecific antibody, for use

-   i) in the modulation of immune responses, such as restoring T cell    activity,-   ii) in stimulating an immune response or function,-   iii) in the treatment of infections,-   iv) in the treatment of cancer,-   v) in delaying progression of cancer,-   vi) in prolonging the survival of a patient suffering from cancer.

In one aspect provided is the bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3 as describedherein, or the pharmaceutical composition comprising the bispecificantibody, for use in the treatment of a disease in an individual in needthereof. In a specific aspect, the invention provides a bispecificantibody comprising a first antigen-binding site that specifically bindsto PD1 and a second antigen-binding site that specifically binds toTIM3, or the pharmaceutical composition comprising the bispecificantibody, for use in the treatment of cancer. In a further specificaspect, a bispecific antibody comprising a first antigen-binding sitethat specifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM3, or the pharmaceutical composition comprisingthe bispecific antibody, for use in the modulation of immune responsesis provided. In another aspect, a bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, or apharmaceutical composition comprising the bispecific antibody for use inthe treatment of a chronic viral infection is provided.

Also provided is the use of the bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3 as described hereinfor the manufacture of a medicament for the treatment of a disease in anindividual in need thereof, in particular for the manufacture of amedicament for the treatment of cancer, as well as a method of treatinga disease in an individual, comprising administering to said individuala therapeutically effective amount of a composition comprising thebispecific antibody comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM3 as described herein in a pharmaceuticallyacceptable form. In a specific aspect, the disease is cancer. In anotherspecific aspect, the disease is a chronic viral infection. In anotheraspect, a method of modulating of immune responses in an individual,comprising administering to said individual a therapeutically effectiveamount of a composition comprising the bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3 as described hereinin a pharmaceutically acceptable form is provided. In any of the aboveaspects the individual is preferably a mammal, particularly a human.

The invention also provides a bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3 as describedherein, or a pharmaceutical composition comprising the bispecificantibody for use in the prevention or treatment of cancer, wherein thebispecific antibody is administered in combination with achemotherapeutic agent, radiation and/or other agents for use in cancerimmunotherapy.

Furthermore, provided is a method of inhibiting the growth of tumorcells in an individual comprising administering to the individual aneffective amount of a bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3 as described hereinto inhibit the growth of the tumor cells. The individual is preferably amammal, particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Blockade of PD1 with chimeric PD1-0103 strongly enhancesIFN-gamma secretion by allogenic stimulated primary human T cells.

FIG. 2: Blockade of PD1 with chimeric PD1-0103 strongly increasesinterferon-gamma (IFN-γ) secretion by allogenic stimulated primary humanT cells.

FIG. 3: Blockade of PD1 with chimeric PD1-0103 strongly increases tumornecrosis factor alpha (TNF) secretion by allogenic stimulated primaryhuman T cells.

FIGS. 4A and 4B: FIG. 4A: frequency of CD4 T cells producing Granzyme B;and FIG. 4B: Amount of IFN-γ detected by absorbance (Optical Density,O.D.) in the supernatant of the MLR in presence of increasingconcentrations of different anti-PD-1 antibodies

FIGS. 5A and 5B: FIG. 5A: Impact of PD1/PD-L1 blockade on reactivationof suppressed T cell receptor signaling in presence of differentanti-PD-1 antibodies; FIG. 5B:) Impact of PD1/PD-L1 blockade onreactivation of suppressed T cell receptor signaling in presence ofdifferent anti-PD-1 antibodies

FIG. 6: Scheme of FRET assay for simultaneous binding of anti-PD1/Tim3bispecific antibodies to recombinant cells

FIGS. 7A and 7B: Induction of FRET upon treatment/binding of differentbispecific PD1TIM3 antibodies on PD1 and TIM3 expressing cells: HEK293cells, double transfected with PD1 SNAP Tim3 CLIP, were stained with 100nM SNAP-Lumi4-Tb (Cisbio) and 100 nM Clip-Red (Cisbio) for 1 h at 370 inTag-Lite buffer (Cisbio). After washing, labelled cells were incubatedwith indicated bispecific anti-PD1/Tim3 antibodies [0-10 nM] for 1 h at4° C. before time-resolved fluorescence was measured at 665/620 nm withan BMG Pherastar reader (depicted is the mean+/−SD of the FRET signal[ratio 665/620 nm*10,000], n=3). FIG. 7A: 1+1 formats (antibodiesPD1TIM3_0389 and PD1TIM3_0168) compared to 2+2 constructs(PD1TIM3_0358+PD1TIM3_0359). FIG. 7B: humanized bispecific variants(PD1TIM3_0476 and PD1TIM3_477).

FIGS. 8A and 8B: FRET assay for simultaneous binding of anti-PD1/TIM3bispecific antibody 1+1 PD1TIM3-0168: SNAP-tagged PD1 and CLIP-taggedTIM3 cells (as described before) were labelled with 100 nM SNAP-Lumi4-Tband 100 nM Clip-Red. After washing, labelled cells were incubated withthe bispecific anti-PD1/TIM3 antibody #0168 [at indicatedconcentrations] for 1 h at 4° C. before time-resolved fluorescence wasmeasured at 665/620 nm with an BMG Pherastar reader (black lines). Tounderline the specificity of the bispecific antibody induced FRETsignal, either an anti-PD1 monoclonal antibody (#0165; FIG. 8A) or ananti-TIM3-blocking antibody (#0018, FIG. 8B) were added for competitionresulting in an almost complete prevention of the FRET signal (greycurves). Treatment with an anti-PD1 antibody alone did not result inFRET induction (dotted lines).

FIG. 9A: Bispecific 1+1 PD1TIM3-0389 shows the same binding ratio topositive CD4+ T-cells (PD1+, TIM3+) than chimeric TIM3_0028 (chi0028)and humanized TIM3-0438 (0438), but less binding to Monocytes, NK cellsand CD3+ T-cells.

FIG. 9B: Bispecific 1+1 PD1TIM3-0389 show significantly increased MFIfor binding to positive CD4+ T-cells (PD1+, TIM3+) than chimericTIM3_0028 (chi0028) and humanized Tim3-0438 (0438).

FIG. 9C: Bispecific 1+1 PD1TIM3-0168: no differences concerning bindingto positive CD4+ T-cells (PD1+, TIM3+) than chimeric TIM3_0018(Tim3-chi0018) and humanized TIM3-0434 (0434).

FIG. 9D: Bispecific 1+1 PD1TIM3-0168 show only slight increased MFI forbinding to positive CD4+ T-cells (PD1+, Tim3+).

FIGS. 9E and 9F: anti-TIM3 antibody TIM3-0038 shows binding to bothmonocytes and CD4+ T-cells.

FIGS. 9G and 9H: Bispecific 1+1 PD1TIM3-0166 (based on chimericPD1-0103/Tim3-0038) shows strongly reduced binding to monocytes(compared to parent anti-TIM3 antibody TIM3_0038 see FIGS. 9E and 9F)while retaining strong binding to CD4+ Tcells.

FIGS. 10A, 10B, 10C and 10D: Bispecific 1+1 PD1TIM3-0166 (based onchimeric PD1-0103/TIM3-0038) showed reduced internalization compared toBispecific 2+2 PD1TIM3-0321 (also based on chimeric PD1-0103/TIM3-0038,but having two antigen binding sites for PD and two for TIM3) andcompared to parent TIM3-0038 antibody on activated CD4+ T-Cells and onactivated NK cells.

FIG. 11A: Analysis over time shows higher membrane localization in bothbispecific and PD1 antibodies when compared to intracellular clusteringof TIM3 antibodies. Antibody designations in Figure: TIM3(chi18-A647=chimeric TIM3_0018 labeled with AlexaA647), a-TIM3(chi28-A647=chimeric TIM3_0028 labeled with AlexaA647), Bispec(0168-A647=1+1 PD1TIM3_0168 (based on chimeric PD1-0103/TIM3-0018)labeled with AlexaA647) Bispec (0389-A647=1+1 PD1TIM3_0389 (based onchimeric PD1-0103/TIM3-0028) labeled with Alexa 647) and a-PD1(0165-A488=chimeric PD1-0103 labeled with Alexa488).

-   -   The anti-PD1 and the bispecific 1+1 PD1TIM3_0389 (Bispec 0389)        show only very slow internalization, even after 3 h, wheras the        internalization for the other bispecific 1+1 PD1TIM3_0168        (Bispec 0168) is stronger. Stronger internalization is shown the        aTIM3 Ab 0028; the most internalization is shown by aTIM3-0018.

FIG. 11B: Chimeric PD1-0103 (aPD1-0165) shows only poor internalization,whereas the high affinity chimeric TIM3_0018 (aTim3-chi18) is stronglyinternalized upon TIM3-binding, even after 15 minutes. Internalizationfor the low affinity binder chimeric TIM3_0028 (aTIM3-chi28) is slightlyreduced. The bispecific 1+1 AB 0168 (composed of high affinity binderaPD1-0165 and high affinity aTIM3-0018) shows more reducedinternalization. The bispecific 1+1 AB 0389 (composed of high affinitybinder chimeric PD1-0103 (aPD1-0165) and low affinity chimeric TIM3_0028(aTIM3-0028) shows very strong reduced internalization. This could bedue to the bivalent binding to PD1 and TIM3, where the high affinitybinding to PD1 retains the antibody at the cell surface.

FIG. 12A: Potency of PD1-TIM3 Bispecific Antibody 1+1 PD1TIM3_0168(based on chimeric PD1-0103/TIM3-0018 (=AB 0168) in comparison withchimeric PD1-0103 (=PD1-0165) and chimeric TIM3_0018 (=TIM3-chi18) andcombinations thereof.

FIG. 12B: Potency of PD1-TIM3 Bispecific Antibody 1+1 PD1TIM3_0389(based on chimeric PD1-0103/TIM3-0028 (=Bispec AB 0389) in comparisonwith chimeric PD1-0103 (=PD1-0165) and chimeric TIM3_0028 (=TIM3-chi28)and combinations thereof.

FIG. 12C: Potency of PD1-TIM3 Bispecific Antibody 1+1 PD1-0103/Ky8213(based on chimeric PD1-0103/and anti-TIM3 Ky8213 from US20120189617 (seeantibody8213 e.g. Example 33) which produced anlalougously as describedin Example 1 as a 1+1 CrossMab) in comparison with chimeric PD1-0103(=PD1-0165) and anti-TIM3-Ky8213 (from US20120189617 (see antibody 8213)e.g. Example 33) and combinations thereof.

FIG. 12D: Potency of PD1-TIM3 Bispecific Antibody 1+1 PD1TIM3_0389(based on chimeric PD1-0103/TIM3-0028 (=Bispec AB 0389 (1+1))) incomparison with PD1-TIM3 Bispecific Antibody 2+2 PD1TIM3_0358 based onchimeric PD1-0103/TIM3-0028 (=Bispec AB 0358 (2+2)), and chimericPD1-0103 (=PD1-0165) and chimeric TIM3_0028 (=TIIM3-chi28) andcombinations thereof.

FIG. 13: Treatment with PD1-TIM3 Bispecific Antibody 1+1 PD1TIM3_0476significantly increased the ability of CD4 T cells to release IFN-gammacompared to treatment with PD1 or TIM3 antibodies alone and evencompared to treatment with a combination of the parent antibody PD1_0376and antibody TIM3_0438. CD4 T cells were co-cultured with aMHCII-expressing tumor cell line. PD1-Tim3 Bispecific Antibody 1+1PD1TIM3_0476 was tested against the PD1 antibodies aPD1_0376, MDX-1106(nivolumab) and MK-3475 (pembrolizumab), against the TIM3 antibodiesaTIM3_0438 and Kyowa-8213 (as disclosed in WO 2011/155697) and againstthe combination of anti-PD1 antibody aPD1-0376 and anti-TIM3 antibodyaTIM3_0438.

FIGS. 14A and 14B: The results of an Efficacy Experiment comparingPD1-TIM3 Bispecific Antibody 1+1 (0476) with PD1 or TIM3 antibodiesalone in immune suppressed female mice (NOG) challenged with MKN45 cellsand provided with PBMC from a healthy human donor are shown in FIGS. 14Aand 14B. The plots represent the measurement mean of tumour size (withina treatment group) including the standard error of the tumour size meanover the period of 30 days. The curves with the filled circle correspondto tumour size growth without treatment (vehicle). In FIG. 14A thetumour growth with lower dose treatment is shown (1.5 mg/kg antibodyPD1_0376, 1.5 mg/kg nivolumab, 1.5 mg/kg antibody TIM3_0438 or 3 mg/kgbispecific antibody 1+1 PD1TIM3_0476); in FIG. 14B the tumor growth athigher doses (5 mg/kg antibody PD1_0376, 5 mg/kg nivolumab, 5 mg/kgantibody Tim3_0438 or 10 mg/kg bispecific antibody 1+1 PD1TIM3_0476) isshown.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as generally used in the art to which thisinvention belongs. For purposes of interpreting this specification, thefollowing definitions will apply and whenever appropriate, terms used inthe singular will also include the plural and vice versa.

As used herein, the term “antigen binding molecule” refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant. Examples of antigen binding molecules are antibodies,antibody fragments and scaffold antigen binding proteins.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, monospecific and multispecificantibodies (e.g., bispecific antibodies), and antibody fragments so longas they exhibit the desired antigen-binding activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g. containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen.

The term “monospecific” antibody as used herein denotes an antibody thathas one or more binding sites each of which bind to the same epitope ofthe same antigen. The term “bispecific” means that the antibody is ableto specifically bind to at least two distinct antigenic determinants,for example two binding sites each formed by a pair of an antibody heavychain variable domain (VH) and an antibody light chain variable domain(VL) binding to different antigens or to different epitopes on the sameantigen. Such a bispecific antibody is an 1+1 format. Other bispecificantibody formats are 2+1 formats (comprising two binding sites for afirst antigen or epitope and one binding site for a second antigen orepitope) or 2+2 formats (comprising two binding sites for a firstantigen or epitope and two binding sites for a second antigen orepitope). Typically, a bispecific antibody comprises two antigen bindingsites, each of which is specific for a different antigenic determinant.

The term “valent” as used within the current application denotes thepresence of a specified number of binding sites in an antigen bindingmolecule. As such, the terms “bivalent”, “tetravalent”, and “hexavalent”denote the presence of two binding sites, four binding sites, and sixbinding sites, respectively, in an antigen binding molecule. Thebispecific antibodies according to the invention are at least “bivalent”and may be “trivalent” or “multivalent” (e.g. “tetravalent” or“hexavalent”). In a particular aspect, the antibodies of the presentinvention have two or more binding sites and are bispecific. That is,the antibodies may be bispecific even in cases where there are more thantwo binding sites (i.e. that the antibody is trivalent or multivalent).In particular, the invention relates to bispecific bivalent antibodies,having one binding site for each antigen they specifically bind to.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure.“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG-classantibodies are heterotetrameric glycoproteins of about 150,000 daltons,composed of two light chains and two heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by alight chain constant domain (CL), also called a light chain constantregion. The heavy chain of an antibody may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ1 (IgG1), γ2 (IgG2),γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies, triabodies, tetrabodies, cross-Fab fragments; linearantibodies; single-chain antibody molecules (e.g. scFv); multispecificantibodies formed from antibody fragments and single domain antibodies.For a review of certain antibody fragments, see Hudson et al., Nat Med9, 129-134 (2003). For a review of scFv fragments, see e.g. Pluckthun,in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg andMoore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion ofFab and F(ab′)2 fragments comprising salvage receptor binding epitoperesidues and having increased in vivo half-life, see U.S. Pat. No.5,869,046. Diabodies are antibody fragments with two antigen-bindingsites that may be bivalent or bispecific, see, for example, EP 404,097;WO 1993/01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollingeret al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies andtetrabodies are also described in Hudson et al., Nat Med 9, 129-134(2003). Single-domain antibodies are antibody fragments comprising allor a portion of the heavy chain variable domain or all or a portion ofthe light chain variable domain of an antibody. In certain embodiments,a single-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see e.g. U.S. Pat. No. 6,248,516 B1). In addition,antibody fragments comprise single chain polypeptides having thecharacteristics of a VH domain, namely being able to assemble togetherwith a VL domain, or of a VL domain, namely being able to assembletogether with a VH domain to a functional antigen binding site andthereby providing the antigen binding property of full lengthantibodies. Antibody fragments can be made by various techniques,including but not limited to proteolytic digestion of an intact antibodyas well as production by recombinant host cells (e.g. E. coli or phage),as described herein.

Papain digestion of intact antibodies produces two identicalantigen-binding fragments, called “Fab” fragments containing each theheavy- and light-chain variable domains and also the constant domain ofthe light chain and the first constant domain (CH1) of the heavy chain.As used herein, Thus, the term “Fab fragment” refers to an antibodyfragment comprising a light chain fragment comprising a VL domain and aconstant domain of a light chain (CL), and a VH domain and a firstconstant domain (CH1) of a heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteins from theantibody hinge region. Fab′-SH are Fab′ fragments wherein the cysteineresidue(s) of the constant domains bear a free thiol group. Pepsintreatment yields an F(ab′)₂ fragment that has two antigen-combiningsites (two Fab fragments) and a part of the Fc region.

The term “cross-Fab fragment” or “xFab fragment” or “crossover Fabfragment” refers to a Fab fragment, wherein either the variable regionsor the constant regions of the heavy and light chain are exchanged. Twodifferent chain compositions of a crossover Fab molecule are possibleand comprised in the bispecific antibodies of the invention: On the onehand, the variable regions of the Fab heavy and light chain areexchanged, i.e. the crossover Fab molecule comprises a peptide chaincomposed of the light chain variable region (VL) and the heavy chainconstant region (CH1), and a peptide chain composed of the heavy chainvariable region (VH) and the light chain constant region (CL). Thiscrossover Fab molecule is also referred to as CrossFab_((VLVH)). On theother hand, when the constant regions of the Fab heavy and light chainare exchanged, the crossover Fab molecule comprises a peptide chaincomposed of the heavy chain variable region (VH) and the light chainconstant region (CL), and a peptide chain composed of the light chainvariable region (VL) and the heavy chain constant region (CH1). Thiscrossover Fab molecule is also referred to as CrossFab_((CLCH1)).

A “single chain Fab fragment” or “scFab” is a polypeptide consisting ofan antibody heavy chain variable domain (VH), an antibody constantdomain 1 (CH1), an antibody light chain variable domain (VL), anantibody light chain constant domain (CL) and a linker, wherein saidantibody domains and said linker have one of the following orders inN-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b)VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL;and wherein said linker is a polypeptide of at least 30 amino acids,preferably between 32 and 50 amino acids. Said single chain Fabfragments are stabilized via the natural disulfide bond between the CLdomain and the CH1 domain. In addition, these single chain Fab moleculesmight be further stabilized by generation of interchain disulfide bondsvia insertion of cysteine residues (e.g. position 44 in the variableheavy chain and position 100 in the variable light chain according toKabat numbering).

A “crossover single chain Fab fragment” or “x-scFab” is a is apolypeptide consisting of an antibody heavy chain variable domain (VH),an antibody constant domain 1 (CH1), an antibody light chain variabledomain (VL), an antibody light chain constant domain (CL) and a linker,wherein said antibody domains and said linker have one of the followingorders in N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 andb) VL-CH1-linker-VH-CL; wherein VH and VL form together anantigen-binding site which binds specifically to an antigen and whereinsaid linker is a polypeptide of at least 30 amino acids. In addition,these x-scFab molecules might be further stabilized by generation ofinterchain disulfide bonds via insertion of cysteine residues (e.g.position 44 in the variable heavy chain and position 100 in the variablelight chain according to Kabat numbering).

A “single-chain variable fragment (scFv)” is a fusion protein of thevariable regions of the heavy (VH) and light chains (V_(L)) of anantibody, connected with a short linker peptide of ten to about 25 aminoacids. The linker is usually rich in glycine for flexibility, as well asserine or threonine for solubility, and can either connect theN-terminus of the VH with the C-terminus of the V_(L), or vice versa.This protein retains the specificity of the original antibody, despiteremoval of the constant regions and the introduction of the linker. scFvantibodies are, e.g. described in Houston, J. S., Methods in Enzymol.203 (1991) 46-96). In addition, antibody fragments comprise single chainpolypeptides having the characteristics of a VH domain, namely beingable to assemble together with a VL domain, or of a VL domain, namelybeing able to assemble together with a VH domain to a functional antigenbinding site and thereby providing the antigen binding property of fulllength antibodies.

“Scaffold antigen binding proteins” are known in the art, for example,fibronectin and designed ankyrin repeat proteins (DARPins) have beenused as alternative scaffolds for antigen-binding domains, see, e.g.,Gebauer and Skerra, Engineered protein scaffolds as next-generationantibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumppet al., Darpins: A new generation of protein therapeutics. DrugDiscovery Today 13: 695-701 (2008). In one aspect of the invention, ascaffold antigen binding protein is selected from the group consistingof CTLA-4 (Evibody), Lipocalins (Anticalin), a Protein A-derivedmolecule such as Z-domain of Protein A (Affibody), an A-domain(Avimer/Maxibody), a serum transferrin (trans-body); a designed ankyrinrepeat protein (DARPin), a variable domain of antibody light chain orheavy chain (single-domain antibody, sdAb), a variable domain ofantibody heavy chain (nanobody, aVH), V_(NAR) fragments, a fibronectin(AdNectin), a C-type lectin domain (Tetranectin); a variable domain of anew antigen receptor beta-lactamase (VNAR fragments), a humangamma-crystallin or ubiquitin (Affilin molecules); a kunitz type domainof human protease inhibitors, microbodies such as the proteins from theknottin family, peptide aptamers and fibronectin (adnectin).

CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-familyreceptor expressed on mainly CD4+ T-cells. Its extracellular domain hasa variable domain-like Ig fold. Loops corresponding to CDRs ofantibodies can be substituted with heterologous sequence to conferdifferent binding properties. CTLA-4 molecules engineered to havedifferent binding specificities are also known as Evibodies (e.g. U.S.Pat. No. 7,166,697B1). Evibodies are around the same size as theisolated variable region of an antibody (e.g. a domain antibody). Forfurther details see Journal of Immunological Methods 248 (1-2), 31-45(2001). Lipocalins are a family of extracellular proteins whichtransport small hydrophobic molecules such as steroids, bilins,retinoids and lipids. They have a rigid beta-sheet secondary structurewith a number of loops at the open end of the conical structure whichcan be engineered to bind to different target antigens. Anticalins arebetween 160-180 amino acids in size, and are derived from lipocalins.For further details see Biochim Biophys Acta 1482: 337-350 (2000), U.S.Pat. No. 7,250,297B1 and US20070224633. An affibody is a scaffoldderived from Protein A of Staphylococcus aureus which can be engineeredto bind to antigen. The domain consists of a three-helical bundle ofapproximately 58 amino acids. Libraries have been generated byrandomization of surface residues. For further details see Protein Eng.Des. Sel. 2004, 17, 455-462 and EP 1641818A1. Avimers are multidomainproteins derived from the A-domain scaffold family. The native domainsof approximately 35 amino acids adopt a defined disulfide bondedstructure. Diversity is generated by shuffling of the natural variationexhibited by the family of A-domains. For further details see NatureBiotechnology 23(12), 1556-1561 (2005) and Expert Opinion onInvestigational Drugs 16(6), 909-917 (June 2007). A transferrin is amonomeric serum transport glycoprotein. Transferrins can be engineeredto bind different target antigens by insertion of peptide sequences in apermissive surface loop. Examples of engineered transferrin scaffoldsinclude the Trans-body. For further details see J. Biol. Chem 274,24066-24073 (1999). Designed Ankyrin Repeat Proteins (DARPins) arederived from Ankyrin which is a family of proteins that mediateattachment of integral membrane proteins to the cytoskeleton. A singleankyrin repeat is a 33 residue motif consisting of two alpha-helices anda beta-turn. They can be engineered to bind different target antigens byrandomizing residues in the first alpha-helix and a beta-turn of eachrepeat. Their binding interface can be increased by increasing thenumber of modules (a method of affinity maturation). For further detailssee J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) andJ. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.

A single-domain antibody is an antibody fragment consisting of a singlemonomeric variable antibody domain. The first single domains werederived from the variable domain of the antibody heavy chain fromcamelids (nanobodies or VHH fragments). Furthermore, the termsingle-domain antibody includes an autonomous human heavy chain variabledomain (aVH) or V_(NAR) fragments derived from sharks. Fibronectin is ascaffold which can be engineered to bind to antigen. Adnectins consistsof a backbone of the natural amino acid sequence of the 10th domain ofthe 15 repeating units of human fibronectin type III (FN3). Three loopsat one end of the .beta.-sandwich can be engineered to enable anAdnectin to specifically recognize a therapeutic target of interest. Forfurther details see Protein Eng. Des. Sel. 18, 435-444 (2005),US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1. Peptideaptamers are combinatorial recognition molecules that consist of aconstant scaffold protein, typically thioredoxin (TrxA) which contains aconstrained variable peptide loop inserted at the active site. Forfurther details see Expert Opin. Biol. Ther. 5, 783-797 (2005).Microbodies are derived from naturally occurring microproteins of 25-50amino acids in length which contain 3-4 cysteine bridges—examples ofmicroproteins include KalataBI and conotoxin and knottins. Themicroproteins have a loop which can beengineered to include upto 25amino acids without affecting the overall fold of the microprotein. Forfurther details of engineered knottin domains, see WO2008098796.

An “antigen binding molecule that binds to the same epitope” as areference molecule refers to an antigen binding molecule that blocksbinding of the reference molecule to its antigen in a competition assayby 50% or more, and conversely, the reference molecule blocks binding ofthe antigen binding molecule to its antigen in a competition assay by50% or more.

As used herein, the term “antigen-binding site” refers to the part ofthe antigen binding molecule that specifically binds to an antigenicdeterminant. More particularly, the term “antigen-binding site” refersthe part of an antibody that comprises the area which specifically bindsto and is complementary to part or all of an antigen. Where an antigenis large, an antigen binding molecule may only bind to a particular partof the antigen, which part is termed an epitope. An antigen-binding sitemay be provided by, for example, one or more variable domains (alsocalled variable regions). Preferably, an antigen-binding site comprisesan antibody light chain variable region (VL) and an antibody heavy chainvariable region (VH). In one aspect, the antigen-binding site is able tobind to its antigen and block or partly block its function.Antigen-binding sites that specifically bind to PD1 or to TIM-3 includeantibodies and fragments thereof as further defined herein. In addition,antigen-binding sites may include scaffold antigen binding proteins,e.g. binding domains which are based on designed repeat proteins ordesigned repeat domains (see e.g. WO 2002/020565).

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, on thesurface of immune cells, free in blood serum, and/or in theextracellular matrix (ECM). The proteins useful as antigens herein canbe any native form the proteins from any vertebrate source, includingmammals such as primates (e.g. humans) and rodents (e.g. mice and rats),unless otherwise indicated. In a particular embodiment the antigen is ahuman protein. Where reference is made to a specific protein herein, theterm encompasses the “full-length”, unprocessed protein as well as anyform of the protein that results from processing in the cell. The termalso encompasses naturally occurring variants of the protein, e.g.splice variants or allelic variants.

By “specific binding” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding molecule to bind to aspecific antigen can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. Surface Plasmon Resonance (SPR) technique (analyzed ona BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)),and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).In one embodiment, the extent of binding of an antigen binding moleculeto an unrelated protein is less than about 10% of the binding of theantigen binding molecule to the antigen as measured, e.g. by SPR. Incertain embodiments, an molecule that binds to the antigen has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁷ M or less, e.g. from 10⁻⁷ M to 10⁻¹³M, e.g. from 10⁻⁹ M to 10⁻¹³ M).

“Affinity” or “binding affinity” refers to the strength of the sum totalof non-covalent interactions between a single binding site of a molecule(e.g. an antibody) and its binding partner (e.g. an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g. antibody and antigen). The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (Kd), which is the ratio of dissociation andassociation rate constants (koff and kon, respectively). Thus,equivalent affinities may comprise different rate constants, as long asthe ratio of the rate constants remains the same. Affinity can bemeasured by common methods known in the art, including those describedherein. A particular method for measuring affinity is Surface PlasmonResonance (SPR).

As used herein, the term “high affinity” of an antibody refers to anantibody having a Kd of 10⁻⁹ M or less and even more particularly 10⁻¹⁰M or less for a target antigen. The term “low affinity” of an antibodyrefers to an antibody having a Kd of 10⁻⁸ or higher.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “a bispecific antibody comprising a first antigen-binding sitethat specifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM-3” “a bispecific antibody that specificallybinds PD1 and TIM-3”, “bispecific antigen binding molecule specific forPD1 and TIM-3” are used interchangeably herein and refer to a bispecificantibody that is capable of binding PD1 and TIM-3 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting PD1 and TIM-3.

The term “PD1”, also known as Programmed cell death protein 1, is a typeI membrane protein of 288 amino acids that was first described in 1992(Ishida et al., EMBO J., 11 (1992), 3887-3895). PD-1 is a member of theextended CD28/CTLA-4 family of T cell regulators and has two ligands,PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The protein's structureincludes an extracellular IgV domain followed by a transmembrane regionand an intracellular tail. The intracellular tail contains twophosphorylation sites located in an immunoreceptor tyrosine-basedinhibitory motif and an immunoreceptor tyrosine-based switch motif,which suggests that PD-1 negatively regulates TCR signals. This isconsistent with binding of SHP-1 and SHP-2 phosphatases to thecytoplasmic tail of PD-1 upon ligand binding. While PD-1 is notexpressed on naïve T cells, it is upregulated following T cell receptor(TCR)-mediated activation and is observed on both activated andexhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772).These exhausted T-cells have a dysfunctional phenotype and are unable torespond appropriately. Although PD-1 has a relatively wide expressionpattern its most important role is likely as a coinhibitory receptor onT cells (Chinai et al, Trends in Pharmacological Sciences 36 (2015),587-595). Current therapeutic approaches thus focus on blocking theinteraction of PD-1 with its ligands to enhance T cell response. Theterms “Programmed Death 1,” “Programmed Cell Death 1,” “Protein PD-1,”“PD-1,” PD1,” “PDCD1,” “hPD-1” and “hPD-I” can be used interchangeably,and include variants, isoforms, species homologs of human PD-1, andanalogs having at least one common epitope with PD-1. The amino acidsequence of human PD1 is shown in UniProt (www.uniprot.org) accessionno. Q15116 (SEQ ID NO:89).

The terms “anti-PD1 antibody” and “an antibody comprising anantigen-binding site that binds to PD1” refer to an antibody that iscapable of binding PD1, especially a PD1 polypeptide expressed on a cellsurface, with sufficient affinity such that the antibody is useful as adiagnostic and/or therapeutic agent in targeting PD1. In one embodiment,the extent of binding of an anti-PD1 antibody to an unrelated, non-PD1protein is less than about 10% of the binding of the antibody to PD1 asmeasured, e.g., by radioimmunoassay (RIA) or flow cytometry (FACS) or bya Surface Plasmon Resonance assay using a biosensor system such as aBiacore® system. In certain embodiments, an antigen binding protein thatbinds to human PD1 has a KD value of the binding affinity for binding tohuman PD1 of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, K 0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10-8 M or less, e.g. from 10-8 M to 10-13 M, e.g., from10-9 M to 10-13 M). In one preferred embodiment the respective KD valueof the binding affinities is determined in a Surface Plasmon Resonanceassay using the Extracellular domain (ECD) of human PD1 (PD1-ECD) forthe PD1 binding affinity. The term “anti-PD1 antibody” also encompassesbispecific antibodies that are capable of binding PD1 and a secondantigen.

The term “TIM3”, the abbreviation for “T cell Immunoglobulin- and Mucindomain-containing molecule 3”, also known as TIM-3, HAVCR2, KIM-3,TIMD3, and FLJ14428, refers to a T helper cell type 1-specific cellsurface protein that regulates macrophage activation and the severity ofinflammatory conditions. TIM3 is also associated with cancer, inparticular, with cancer stem cells. The nucleotide and protein sequencesof TIM3 are known for many species. For example, the human amino acidsequence can be found under Uniprot accession number Q8TDQ0 (SEQ IDNO:93). The human protein is characterized by an extracellular domaincomprising an Ig like domain and a mucin domain (further comprisingO-linked and N-linked glycosylation sites) comprising approximatelyamino acids 22-202, a transmembrane domain (amino acids 203-223), and anintracellular (cytoplasmic) domain (amino acids 224-301). For the humanTIM3 protein shown as SEQ ID NO: 93, the extracellular domain comprisesapproximately amino acids 22-202, the transmembrane domain comprisesapproximately amino acids 203-223, and the cytoplasmic domain comprisesapproximately amino acids 224-301. The term “TIM3” includes variants,isoforms, species homologs of human TIM3, and analogs having at leastone common epitope with TIM3.

The terms “anti-TIM3 antibody” and “an antibody comprising anantigen-binding site that binds to TIM3” refer to an antibody that iscapable of binding TIM3, especially a TIM3 polypeptide expressed on acell surface, with sufficient affinity such that the antibody is usefulas a diagnostic and/or therapeutic agent in targeting TIM3. In oneembodiment, the extent of binding of an anti-TIM3 antibody to anunrelated, non-TIM3 protein is less than about 10% of the binding of theantibody to TIM3 as measured, e.g., by radioimmunoassay (RIA) or flowcytometry (FACS) or by a Surface Plasmon Resonance assay using abiosensor system such as a Biacore® system. In certain embodiments, anantigen binding protein that binds to human TIM3 has a KD value of thebinding affinity for binding to human TIM3 of ≤1 μM, 100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10-8 M or less, e.g. from 10-7M to 10-13 M, e.g., from 10-9 M to 10-13 M). In one preferred embodimentthe respective KD value of the binding affinities is determined in aSurface Plasmon Resonance assay using the Extracellular domain (ECD) ofhuman TIM3 (TIM3-ECD) for the TIM3 binding affinity. The term “anti-TIM3antibody” also encompasses bispecific antibodies that are capable ofbinding TIM3 and a second antigen.

A “blocking” antibody or an “antagonist” antibody is one that inhibitsor reduces a biological activity of the antigen it binds. In someembodiments, blocking antibodies or antagonist antibodies substantiallyor completely inhibit the biological activity of the antigen. Forexample, the bispecific antibodies of the invention block the signalingthrough PD-1 and TIM-3 so as to restore a functional response by T cells(e.g., proliferation, cytokine production, target cell killing) from adysfunctional state to antigen stimulation.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding the antigenbinding molecule to antigen. The variable domains of the heavy chain andlight chain (VH and VL, respectively) of a native antibody generallyhave similar structures, with each domain comprising four conservedframework regions (FRs) and three hypervariable regions (HVRs). See,e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page91 (2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR,” as used herein refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.

Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) Hypervariable regions(HVRs) are also referred to as complementarity determining regions(CDRs), and these terms are used herein interchangeably in reference toportions of the variable region that form the antigen binding regions.This particular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, “Sequences of Proteins of ImmunologicalInterest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917(1987), where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table A as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE A CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table A isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²″AbM″ with a lowercase “b” as used in Table A refers to theCDRs as defined by Oxford Molecular's ″AbM″ antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. CDRs also comprise“specificity determining residues,” or “SDRs,” which are residues thatcontact antigen. SDRs are contained within regions of the CDRs calledabbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2,a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633(2008).) Unless otherwise indicated, HVR residues and other residues inthe variable domain (e.g., FR residues) are numbered herein according toKabat et al.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g. IgG₁, IgG2,IgG3, IgG4, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ respectively.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization. Other forms of “humanized antibodies” encompassed by thepresent invention are those in which the constant region has beenadditionally modified or changed from that of the original antibody togenerate the properties according to the invention, especially in regardto C1q binding and/or Fc receptor (FcR) binding.

A “human” antibody is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an antibody heavy chain that contains at least aportion of the constant region. The term includes native sequence Fcregions and variant Fc regions. Particularly, a human IgG heavy chain Fcregion extends from Cys226, or from Pro230, to the carboxyl-terminus ofthe heavy chain. However, the C-terminal lysine (Lys447) of the Fcregion may or may not be present. The amino acid sequences of the heavychains are always presented with the C-terminal lysine, however variantswithout the C-terminal lysine are included in the invention.

An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain. The “CH2domain” of a human IgG Fc region usually extends from an amino acidresidue at about position 231 to an amino acid residue at about position340. In one embodiment, a carbohydrate chain is attached to the CH2domain. The CH2 domain herein may be a native sequence CH2 domain orvariant CH2 domain. The “CH3 domain” comprises the stretch of residuesC-terminal to a CH2 domain in an Fc region (i.e. from an amino acidresidue at about position 341 to an amino acid residue at about position447 of an IgG). The CH3 region herein may be a native sequence CH3domain or a variant CH3 domain (e.g. a CH3 domain with an introduced“protuberance” (“knob”) in one chain thereof and a correspondingintroduced “cavity” (“hole”) in the other chain thereof; see U.S. Pat.No. 5,821,333, expressly incorporated herein by reference). Such variantCH3 domains may be used to promote heterodimerization of twonon-identical antibody heavy chains as herein described. Unlessotherwise specified herein, numbering of amino acid residues in the Fcregion or constant region is according to the EU numbering system, alsocalled the EU index, as described in Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991.

The “knob-into-hole” technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). The protuberance and cavitycan be made by altering the nucleic acid encoding the polypeptides, e.g.by site-specific mutagenesis, or by peptide synthesis. In a specificembodiment a knob modification comprises the amino acid substitutionT366W in one of the two subunits of the Fc domain, and the holemodification comprises the amino acid substitutions T366S, L368A andY407V in the other one of the two subunits of the Fc domain. In afurther specific embodiment, the subunit of the Fc domain comprising theknob modification additionally comprises the amino acid substitutionS354C, and the subunit of the Fc domain comprising the hole modificationadditionally comprises the amino acid substitution Y349C. Introductionof these two cysteine residues results in the formation of a disulfidebridge between the two subunits of the Fc region, thus furtherstabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

A “region equivalent to the Fc region of an immunoglobulin” is intendedto include naturally occurring allelic variants of the Fc region of animmunoglobulin as well as variants having alterations which producesubstitutions, additions, or deletions but which do not decreasesubstantially the ability of the immunoglobulin to mediate effectorfunctions (such as antibody-dependent cellular cytotoxicity). Forexample, one or more amino acids can be deleted from the N-terminus orC-terminus of the Fc region of an immunoglobulin without substantialloss of biological function. Such variants can be selected according togeneral rules known in the art so as to have minimal effect on activity(see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)).

The term “effector functions” refers to those biological activitiesattributable to the Fc region of an antibody, which vary with theantibody isotype. Examples of antibody effector functions include: C1qbinding and complement dependent cytotoxicity (CDC), Fc receptorbinding, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune complex-mediated antigen uptake by antigen presenting cells, downregulation of cell surface receptors (e.g. B cell receptor), and B cellactivation.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89). A particular activating Fc receptor is human FcγRIIIa (seeUniProt accession no. P08637, version 141).

The term “peptide linker” refers to a peptide comprising one or moreamino acids, typically about 2 to 20 amino acids. Peptide linkers areknown in the art or are described herein. Suitable, non-immunogeniclinker peptides are, for example, (G₄S)_(n), (SG₄)_(n) or G₄(SG₄)_(n)peptide linkers, wherein “n” is generally a number between 1 and 10,typically between 2 and 4, in particular 2.

By “fused” or “connected” is meant that the components (e.g. anantigen-binding site and a FC domain) are linked by peptide bonds,either directly or via one or more peptide linkers.

The term “amino acid” as used within this application denotes the groupof naturally occurring carboxy α-amino acids comprising alanine (threeletter code: ala, one letter code: A), arginine (arg, R), asparagine(asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q),glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine(ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M),phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine(thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide (protein) sequence is defined as the percentage of aminoacid residues in a candidate sequence that are identical with the aminoacid residues in the reference polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN. SAWIor Megalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for aligning sequences, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2. The ALIGN-2 sequence comparison computerprogram was authored by Genentech, Inc., and the source code has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available from Genentech,Inc., South San Francisco, Calif., or may be compiled from the sourcecode. The ALIGN-2 program should be compiled for use on a UNIX operatingsystem, including digital UNIX V4.0D. All sequence comparison parametersare set by the ALIGN-2 program and do not vary. In situations whereALIGN-2 is employed for amino acid sequence comparisons, the % aminoacid sequence identity of a given amino acid sequence A to, with, oragainst a given amino acid sequence B (which can alternatively bephrased as a given amino acid sequence A that has or comprises a certain% amino acid sequence identity to, with, or against a given amino acidsequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

In certain aspects, amino acid sequence variants of the bispecificantibodies of the invention provided herein are contemplated. Forexample, it may be desirable to improve the binding affinity and/orother biological properties of the bispecific antibodies. Amino acidsequence variants of the bispecific antibodies may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the molecules, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.Sites of interest for substitutional mutagenesis include the HVRs andFramework (FRs). Conservative substitutions are provided in Table Bunder the heading “Preferred Substitutions” and further described belowin reference to amino acid side chain classes (1) to (6). Amino acidsubstitutions may be introduced into the molecule of interest and theproducts screened for a desired activity, e.g., retained/improvedantigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE B Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, le;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “amino acid sequence variants” includes substantial variantswherein there are amino acid substitutions in one or more hypervariableregion residues of a parent antigen binding molecule (e.g. a humanizedor human antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antigen binding molecule and/or will havesubstantially retained certain biological properties of the parentantigen binding molecule. An exemplary substitutional variant is anaffinity matured antibody, which may be conveniently generated, e.g.,using phage display-based affinity maturation techniques such as thosedescribed herein. Briefly, one or more HVR residues are mutated and thevariant antigen binding molecules displayed on phage and screened for aparticular biological activity (e.g. binding affinity). In certainembodiments, substitutions, insertions, or deletions may occur withinone or more HVRs so long as such alterations do not substantially reducethe ability of the antigen binding molecule to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. A useful method for identification of residues orregions of an antibody that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and Wells(1989) Science, 244:1081-1085. In this method, a residue or group oftarget residues (e.g., charged residues such as Arg, Asp, His, Lys, andGlu) are identified and replaced by a neutral or negatively chargedamino acid (e.g., alanine or polyalanine) to determine whether theinteraction of the antibody with antigen is affected. Furthersubstitutions may be introduced at the amino acid locationsdemonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of anantigen-antigen binding molecule complex to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues may be targeted or eliminated as candidates for substitution.Variants may be screened to determine whether they contain the desiredproperties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includebispecific antibodies with an N-terminal methionyl residue. Otherinsertional variants of the molecule include the fusion to the N- orC-terminus to a polypeptide which increases the serum half-life of thebispecific antibody.

In certain aspects, the bispecific antibodies provided herein arealtered to increase or decrease the extent to which the antibody isglycosylated. Glycosylation variants of the molecules may beconveniently obtained by altering the amino acid sequence such that oneor more glycosylation sites is created or removed, e.g. thecarbohydrates attached to the Fc domain may be altered. Nativeantibodies produced by mammalian cells typically comprise a branched,biantennary oligosaccharide that is generally attached by an N-linkageto Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.TIBTECH 15:26-32 (1997). The oligosaccharide may include variouscarbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose,and sialic acid, as well as a fucose attached to a GlcNAc in the “stem”of the biantennary oligosaccharide structure. In some embodiments,modifications of the oligosaccharide in the bispecific antibodies of theinvention may be made in order to create variants with certain improvedproperties. In one aspect, variants of bispecific antibodies areprovided having a carbohydrate structure that lacks fucose attached(directly or indirectly) to an Fc region. Such fucosylation variants mayhave improved ADCC function, see e.g. US Patent Publication Nos. US2003/0157108 (Presta, L.) or US 2004/0093621 (Kyowa Hakko Kogyo Co.,Ltd). Further variants of the bispecific antibodies of the inventioninclude those with bisected oligosaccharides, e.g., in which abiantennary oligosaccharide attached to the Fc region is bisected byGlcNAc. Such variants may have reduced fucosylation and/or improved ADCCfunction, see for example WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).Variants with at least one galactose residue in the oligosaccharideattached to the Fc region are also provided. Such antibody variants mayhave improved CDC function and are described, e.g., in WO 1997/30087(Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

In certain aspects, it may be desirable to create cysteine engineeredvariants of the bispecific antibodies of the invention, e.g.,“thioMAbs,” in which one or more residues of the molecule aresubstituted with cysteine residues. In particular embodiments, thesubstituted residues occur at accessible sites of the molecule. Bysubstituting those residues with cysteine, reactive thiol groups arethereby positioned at accessible sites of the antibody and may be usedto conjugate the antibody to other moieties, such as drug moieties orlinker-drug moieties, to create an immunoconjugate. In certainembodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of theheavy chain Fc region. Cysteine engineered antigen binding molecules maybe generated as described, e.g., in U.S. Pat. No. 7,521,541.

In certain aspects, the bispecific antibodies provided herein may befurther modified to contain additional non-proteinaceous moieties thatare known in the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer isattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether thebispecific antibody derivative will be used in a therapy under definedconditions, etc.

In another aspect, conjugates of an antibody and non-proteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the non-proteinaceous moiety is a carbonnanotube (Kam, N. W. et al., Proc. Natl. Acad. Sci. USA 102 (2005)11600-11605). The radiation may be of any wavelength, and includes, butis not limited to, wavelengths that do not harm ordinary cells, butwhich heat the non-proteinaceous moiety to a temperature at which cellsproximal to the antibody-non-proteinaceous moiety are killed.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding apolypeptide contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode bispecific antigen bindingmolecules of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode bispecific antigen binding molecules of the invention orfragments thereof.

The terms “host cell”, “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe bispecific antigen binding molecules of the present invention. Inparticular, the host cell is a prokaryotic or eukaryotic host cell. Hostcells include cultured cells, e.g. mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable excipient” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable excipient includes,but is not limited to, a buffer, a stabilizer, or a preservative.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, the moleculesof the invention are used to delay development of a disease or to slowthe progression of a disease.

The term “cancer” as used herein refers to proliferative diseases, suchas lymphomas, lymphocytic leukemias, lung cancer, non-small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, uterine cancer, ovarian cancer, rectal cancer,cancer of the anal region, stomach cancer, gastric cancer, colon cancer,breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, prostate cancer, cancer of the bladder,cancer of the kidney or ureter, renal cell carcinoma, carcinoma of therenal pelvis, mesothelioma, hepatocellular cancer, biliary cancer,neoplasms of the central nervous system (CNS), spinal axis tumors, brainstem glioma, glioblastoma multiforme, astrocytomas, schwanomas,ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas,pituitary adenoma and Ewings sarcoma, including refractory versions ofany of the above cancers, or a combination of one or more of the abovecancers.

Bispecific Antibodies of the Invention

The invention provides novel bispecific antibodies comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM-3, with particularlyadvantageous properties such as producibility, stability, bindingaffinity, biological activity, specific targeting of certain T cells,targeting efficiency and reduced toxicity. In particular, these arebispecific antibodies, wherein the bispecific antibody binds to PD1 withhigh affinity and to TIM3 with low affinity.

A. Exemplary Bispecific Antibodies that Bind to PD1 and TIM-3

In one aspect, the invention provides a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein

-   -   said first antigen-binding site specifically binding to PD1        comprises        -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:37,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:38, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:39; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:40;        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:41, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:42; and    -   said second antigen-binding site specifically binding to TIM3        comprises    -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:1,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:2, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:3; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4            or SEQ ID NO:11 or SEQ ID NO:12,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:5, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:6; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:17,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:18, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:19; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:20,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:21, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:22; or    -   (c) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:29,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:30, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:31; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:32,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:33, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:34.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein

-   -   said first antigen-binding site specifically binding to PD1        comprises        -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:37,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:38, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:39; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:40;        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:41, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:42; and        -   said second antigen-binding site specifically binding to            TIM3 comprises        -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:1,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:2, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:3; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:12,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:5, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:6.

In another particular aspect, provided is a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,wherein

-   -   said first antigen-binding site specifically binding to PD1        comprises        -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:37,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:38, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:39; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:40;        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:41, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:42; and        -   said second antigen-binding site specifically binding to            TIM3 comprises        -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:17,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:18, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:19; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:20,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:21, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:22.

In a further aspect, provided is a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein

-   -   said first antigen-binding site specifically binding to PD1        comprises        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 43 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 44, or        -   (b) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 46, or        -   (c) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 47, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 48, or        -   (e) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 49,    -   and said second antigen-binding site specifically binding to        TIM3 comprises        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 7 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 8, or        -   (b) a VH domain comprising the amino acid sequence of SEQ ID            NO: 9 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 10, or        -   (c) a VH domain comprising the amino acid sequence of SEQ ID            NO: 13 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 14, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 15 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 16, or        -   (e) a VH domain comprising the amino acid sequence of SEQ ID            NO: 23 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 24, or        -   (f) a VH domain comprising the amino acid sequence of SEQ ID            NO: 25 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 26, or        -   (g) a VH domain comprising the amino acid sequence of SEQ ID            NO: 27 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 28, or        -   (h) a VH domain comprising the amino acid sequence of SEQ ID            NO: 35 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 36.

In a further aspect, the bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3 is a human,humanized or chimeric antibody. In particular, it is a humanizedantibody.

In one aspect, provided is a humanized, bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein

-   -   said first antigen-binding site specifically binding to PD1        comprises        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 46, or        -   (b) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 47, or        -   (c) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 48, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 49,    -   and said second antigen-binding site specifically binding to        TIM3 comprises        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 13 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 14, or        -   (b) a VH domain comprising the amino acid sequence of SEQ ID            NO: 15 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 16, or        -   (c) a VH domain comprising the amino acid sequence of SEQ ID            NO: 25 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 26, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 27 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 28.

In a particular aspect, provided is a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein

-   -   said first antigen-binding site specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 45 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 46,    -   and said second antigen-binding site specifically binding to        TIM3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 15 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 16 or a VH domain comprising the amino acid        sequence of SEQ ID NO: 25 and a VL domain comprising the amino        acid sequence of SEQ ID NO: 26.

Particularly, the invention provides a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein

-   -   said first antigen-binding site specifically binding to PD1        comprises        -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:37,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:38, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:39; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:40;        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:41, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:42; and    -   said second antigen-binding site specifically binding to TIM3        comprises a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:17,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:18, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:19; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:20,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:21, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:22.

More particularly, provided is a bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein said firstantigen-binding site specifically binding to PD1 comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 45 and a VL domaincomprising the amino acid sequence selected from the group consisting ofSEQ ID NO: 46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, and saidsecond antigen-binding site specifically binding to TIM3 comprises a VHdomain comprising the amino acid sequence of SEQ ID NO: 25 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 26.

More specifically, provided is a bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein said firstantigen-binding site specifically binding to PD1 comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 45 and a VL domaincomprising the amino acid sequence of SEQ ID NO: 46, and said secondantigen-binding site specifically binding to TIM3 comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 25 and a VL domaincomprising the amino acid sequence of SEQ ID NO: 26.

In a further aspect, provided is a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein said firstantigen-binding site specifically binding to PD1 comprises a VH domaincomprising the amino acid sequence of SEQ ID NO: 45 and a VL domaincomprising the amino acid sequence selected from the group consisting ofSEQ ID NO: 46, SEQ ID NO:47, SEQ ID NO:48 and SEQ ID NO:49, and saidsecond antigen-binding site specifically binding to TIM3 comprises a VHdomain comprising the amino acid sequence of SEQ ID NO: 27 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 28.

In one aspect, the bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3 is bivalent. Thismeans that the bispecific antibody comprises one antigen-binding sitethat specifically binds to PD1 and one antigen-binding site thatspecifically binds to TIM3 (1+1 format).

In another aspect, provided is a bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein thebispecific antibody binds to TIM3 with low affinity and binds to PD1with high affinity. In a particular aspect, the invention provides abispecific antibody comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM3, wherein the bispecific antibody binds toTIM3 with an at least 50 fold lower binding affinity when compared tothe binding to PD1, more particularly with an at least 100 fold lowerbinding affinity when compared to the binding to PD1. In one preferredembodiment the binding affinity (KD) is determined with Surface PlasmonResonance Assay (as described e.g. in Example 12.)

In one aspect, thus provided is a bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein

-   -   said first antigen-binding site specifically binding to PD1 with        high affinity comprises        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 43 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 44, or        -   (b) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 46, or        -   (c) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 47, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 48, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 49,    -   and said second antigen-binding site specifically binding to        TIM3 with low affinity comprises        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 23 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 24, or        -   (c) a VH domain comprising the amino acid sequence of SEQ ID            NO: 25 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 26, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 27 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 28.

In a specific aspect, provided is a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein said firstantigen-binding site specifically binding to PD1 with high affinitycomprises a VH domain comprising the amino acid sequence of SEQ ID NO:45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46,and said second antigen-binding site specifically binding to TIM3 withlow affinity comprises a VH domain comprising the amino acid sequence ofSEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQID NO: 26.

In another aspect, the invention relates to a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,wherein the bispecific antibody comprises an Fc domain, a first Fabfragment comprising the antigen-binding site that specifically binds toPD1 and a second Fab fragment comprising the antigen-binding site thatspecifically binds to TIM3.

In particular, the Fc domain is an IgG domain, more particularly an IgG1Fc domain or an IgG4 Fc domain.

In another aspect, the invention provides a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,comprising

-   (a) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 50, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 52, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 51, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:53, or-   (b) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 54, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 56, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 55, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:57, or-   (c) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 58, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 60, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 59, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:61, or-   (d) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 62, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 64, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 63, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:65, or-   (e) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 66, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 68, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 67, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:69.

In a particular aspect, the invention provides a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,comprising

-   (a) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 50, a first light chain comprising the amino acid sequence of    SEQ ID NO: 52, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 51, and a second light chain comprising the    amino acid sequence of SEQ ID NO:53, or-   (b) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 54, a first light chain comprising the amino acid sequence of    SEQ ID NO: 56, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 55, and a second light chain comprising the    amino acid sequence of SEQ ID NO:57, or-   (c) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 58, a first light chain comprising the amino acid sequence of    SEQ ID NO: 60, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 59, and a second light chain comprising the    amino acid sequence of SEQ ID NO:61, or-   (d) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 62, a first light chain comprising the amino acid sequence of    SEQ ID NO: 64, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 63, and a second light chain comprising the    amino acid sequence of SEQ ID NO:65, or-   (e) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 66, a first light chain comprising the amino acid sequence of    SEQ ID NO: 68, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 67, and a second light chain comprising the    amino acid sequence of SEQ ID NO:69.

More particularly, the invention provides a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,comprising a first heavy chain comprising the amino acid sequence of SEQID NO: 62, a first light chain comprising the amino acid sequence of SEQID NO: 64, a second heavy chain comprising the amino acid sequence ofSEQ ID NO: 63, and a second light chain comprising the amino acidsequence of SEQ ID NO:65.

In another particular aspect, provided is a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3, afirst heavy chain comprising the amino acid sequence of SEQ ID NO: 66, afirst light chain comprising the amino acid sequence of SEQ ID NO: 68, asecond heavy chain comprising the amino acid sequence of SEQ ID NO: 67,and a second light chain comprising the amino acid sequence of SEQ IDNO:69.

In another aspect, the bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3 is tetravalent. Inone aspect, the bispecific antibody comprises two antigen-binding sitesthat specifically bind to PD1 and two antigen-binding sites thatspecifically bind to TIM3 (2+2 format).

In one aspect, the bispecific antibody of the invention comprises

(a) two light chains and two heavy chains of an antibody comprising twoFab fragments comprising the antigen-binding sites that specificallybind to TIM3, and(b) two additional Fab fragments comprising the antigen-binding sitesthat specifically bind to PD1, wherein said additional Fab fragments areeach connected via a peptide linker to the C-terminus of the heavychains of (a).

In a particular aspect, the peptide linker is (G4S)₄. In another aspect,the two additional Fab fragments comprising the antigen-binding sitesthat specifically bind to PD1 are crossover Fab fragments wherein thevariable domains VL and VH are replaced by each other and the VL-CHchains are each connected via a peptide linker to the C-terminus of theheavy chains of (a).

In a particular aspect, the invention provides a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3,comprising

-   (a) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO: 70, a first light chain comprising the amino acid sequence of    SEQ ID NO: 71, and a second light chain comprising the amino acid    sequence of SEQ ID NO:72, or-   (b) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO: 73, a first light chain comprising the amino acid sequence of    SEQ ID NO: 74, and a second light chain comprising the amino acid    sequence of SEQ ID NO:75, or-   (c) two heavy chains, each comprising the amino acid sequence of SEQ    ID NO: 76, a first light chain comprising the amino acid sequence of    SEQ ID NO: 77, and a second light chain comprising the amino acid    sequence of SEQ ID NO:78.

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

In certain aspects, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

The following section describes preferred aspects of the bispecificantigen binding molecules of the invention comprising Fc domainmodifications reducing Fc receptor binding and/or effector function. Inone aspect, the invention relates to a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM3, wherein the Fcdomain comprises one or more amino acid substitution that reducesbinding to an Fc receptor, in particular towards Fcγ receptor. Inparticular, the Fc domain is of human IgG1 subclass with the amino acidmutations L234A, L235A and P329G (numbering according to Kabat EUindex).

The Fc domain confers favorable pharmacokinetic properties to thebispecific antibodies of the invention, including a long serum half-lifewhich contributes to good accumulation in the target tissue and afavorable tissue-blood distribution ratio. At the same time it may,however, lead to undesirable targeting of the bispecific antibodies ofthe invention to cells expressing Fc receptors rather than to thepreferred antigen-bearing cells. Accordingly, in particular embodimentsthe Fc domain of the bispecific antibodies of the invention exhibitsreduced binding affinity to an Fc receptor and/or reduced effectorfunction, as compared to a native IgG Fc domain, in particular an IgG1FC domain or an IgG4 Fc domain. More particularly, the Fc domain is anIgG1 FC domain.

In one such aspect the Fc domain (or the bispecific antigen bindingmolecule of the invention comprising said Fc domain) exhibits less than50%, preferably less than 20%, more preferably less than 10% and mostpreferably less than 5% of the binding affinity to an Fc receptor, ascompared to a native IgG1 Fc domain (or the bispecific antigen bindingmolecule of the invention comprising a native IgG1 Fc domain), and/orless than 50%, preferably less than 20%, more preferably less than 10%and most preferably less than 5% of the effector function, as comparedto a native IgG1 Fc domain (or the bispecific antigen binding moleculeof the invention comprising a native IgG1 Fc domain). In one aspect, theFc domain (or the bispecific antigen binding molecule of the inventioncomprising said Fc domain) does not substantially bind to an Fc receptorand/or induce effector function. In a particular aspect the Fc receptoris an Fcγ receptor. In one aspect, the Fc receptor is a human Fcreceptor. In one aspect, the Fc receptor is an activating Fc receptor.In a specific aspect, the Fc receptor is an activating human Fcγreceptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, mostspecifically human FcγRIIIa. In one aspect, the Fc receptor is aninhibitory Fc receptor. In a specific aspect, the Fc receptor is aninhibitory human Fcγ receptor, more specifically human FcγRIIB. In oneaspect the effector function is one or more of CDC, ADCC, ADCP, andcytokine secretion. In a particular aspect, the effector function isADCC. In one aspect, the Fc domain exhibits substantially similarbinding affinity to neonatal Fc receptor (FcRn), as compared to a nativeIgG1 Fc domain. Substantially similar binding to FcRn is achieved whenthe Fc domain (or the bispecific antigen binding molecule of theinvention comprising said Fc domain) exhibits greater than about 70%,particularly greater than about 80%, more particularly greater thanabout 90% of the binding affinity of a native IgG1 Fc domain (or thebispecific antigen binding molecule of the invention comprising a nativeIgG1 Fc domain) to FcRn.

In a particular aspect, the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In a particular aspect, the Fcdomain of the bispecific antigen binding molecule of the inventioncomprises one or more amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function.Typically, the same one or more amino acid mutation is present in eachof the two subunits of the Fc domain. In one aspect, the amino acidmutation reduces the binding affinity of the Fc domain to an Fcreceptor. In another aspect, the amino acid mutation reduces the bindingaffinity of the Fc domain to an Fc receptor by at least 2-fold, at least5-fold, or at least 10-fold. In one aspect, the bispecific antigenbinding molecule of the invention comprising an engineered Fc domainexhibits less than 20%, particularly less than 10%, more particularlyless than 5% of the binding affinity to an Fc receptor as compared tobispecific antibodies of the invention comprising a non-engineered Fcdomain. In a particular aspect, the Fc receptor is an Fcγ receptor. Inother aspects, the Fc receptor is a human Fc receptor. In one aspect,the Fc receptor is an inhibitory Fc receptor. In a specific aspect, theFc receptor is an inhibitory human Fcγ receptor, more specifically humanFcγRIIB. In some aspects the Fc receptor is an activating Fc receptor.In a specific aspect, the Fc receptor is an activating human Fcγreceptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, mostspecifically human FcγRIIIa. Preferably, binding to each of thesereceptors is reduced. In some aspects, binding affinity to a complementcomponent, specifically binding affinity to C1q, is also reduced. In oneaspect, binding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e. preservation of the bindingaffinity of the Fc domain to said receptor, is achieved when the Fcdomain (or the bispecific antigen binding molecule of the inventioncomprising said Fc domain) exhibits greater than about 70% of thebinding affinity of a non-engineered form of the Fc domain (or thebispecific antigen binding molecule of the invention comprising saidnon-engineered form of the Fc domain) to FcRn. The Fc domain, or thebispecific antigen binding molecule of the invention comprising said Fcdomain, may exhibit greater than about 80% and even greater than about90% of such affinity. In certain embodiments the Fc domain of thebispecific antigen binding molecule of the invention is engineered tohave reduced effector function, as compared to a non-engineered Fcdomain. The reduced effector function can include, but is not limitedto, one or more of the following: reduced complement dependentcytotoxicity (CDC), reduced antibody-dependent cell-mediatedcytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis(ADCP), reduced cytokine secretion, reduced immune complex-mediatedantigen uptake by antigen-presenting cells, reduced binding to NK cells,reduced binding to macrophages, reduced binding to monocytes, reducedbinding to polymorphonuclear cells, reduced direct signaling inducingapoptosis, reduced dendritic cell maturation, or reduced T cell priming.

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581). Certain antibody variants with improved or diminishedbinding to FcRs are described. (e.g. U.S. Pat. No. 6,737,056; WO2004/056312, and Shields, R. L. et al., J. Biol. Chem. 276 (2001)6591-6604).

In one aspect of the invention, the Fc domain comprises an amino acidsubstitution at a position of E233, L234, L235, N297, P331 and P329. Insome aspects, the Fc domain comprises the amino acid substitutions L234Aand L235A (“LALA”). In one such embodiment, the Fc domain is an IgG1 Fcdomain, particularly a human IgG1 Fc domain. In one aspect, the Fcdomain comprises an amino acid substitution at position P329. In a morespecific aspect, the amino acid substitution is P329A or P329G,particularly P329G. In one embodiment the Fc domain comprises an aminoacid substitution at position P329 and a further amino acid substitutionselected from the group consisting of E233P, L234A, L235A, L235E, N297A,N297D or P331S. In more particular embodiments the Fc domain comprisesthe amino acid mutations L234A, L235A and P329G (“P329G LALA”). The“P329G LALA” combination of amino acid substitutions almost completelyabolishes Fcγ receptor binding of a human IgG1 Fc domain, as describedin PCT Patent Application No. WO 2012/130831 A1. Said document alsodescribes methods of preparing such mutant Fc domains and methods fordetermining its properties such as Fc receptor binding or effectorfunctions. such antibody is an IgG1 with mutations L234A and L235A orwith mutations L234A, L235A and P329G (numbering according to EU indexof Kabat et al, Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991).

In one aspect, the bispecific antibody of the invention comprises (allpositions according to EU index of Kabat) (i) a homodimeric Fc-region ofthe human IgG1 subclass optionally with the mutations P329G, L234A andL235A, or (ii) a homodimeric Fc-region of the human IgG4 subclassoptionally with the mutations P329G, S228P and L235E, or (iii) ahomodimeric Fc-region of the human IgG1 subclass optionally with themutations P329G, L234A, L235A, 1253A, H310A, and H435A, or optionallywith the mutations P329G, L234A, L235A, H310A, H433A, and Y436A, or (iv)a heterodimeric Fc-region wherein one Fc-region polypeptide comprisesthe mutation T366W, and the other Fc-region polypeptide comprises themutations T366S, L368A and Y407V, or wherein one Fc-region polypeptidecomprises the mutations T366W and Y349C, and the other Fc-regionpolypeptide comprises the mutations T366S, L368A, Y407V, and S354C, orwherein one Fc-region polypeptide comprises the mutations T366W andS354C, and the other Fc-region polypeptide comprises the mutationsT366S, L368A, Y407V and Y349C, or (v) a heterodimeric Fc-region of thehuman IgG1 subclass wherein both Fc-region polypeptides comprise themutations P329G, L234A and L235A and one Fc-region polypeptide comprisesthe mutation T366W, and the other Fc-region polypeptide comprises themutations T366S, L368A and Y407V, or wherein one Fc-region polypeptidecomprises the mutations T366W and Y349C, and the other Fc-regionpolypeptide comprises the mutations T366S, L368A, Y407V, and S354C, orwherein one Fc-region polypeptide comprises the mutations T366W andS354C, and the other Fc-region polypeptide comprises the mutationsT366S, L368A, Y407V and Y349C.

In one aspect, the Fc domain is an IgG4 Fc domain. In a more specificembodiment, the Fc domain is an IgG4 Fc domain comprising an amino acidsubstitution at position S228 (Kabat numbering), particularly the aminoacid substitution S228P. In a more specific embodiment, the Fc domain isan IgG4 Fc domain comprising amino acid substitutions L235E and S228Pand P329G. This amino acid substitution reduces in vivo Fab arm exchangeof IgG4 antibodies (see Stubenrauch et al., Drug Metabolism andDisposition 38, 84-91 (2010)). Thus, in one aspect, provided is abispecific antibody, comprising (all positions according to EU index ofKabat) a heterodimeric Fc-region of the human IgG4 subclass wherein bothFc-region polypeptides comprise the mutations P329G, S228P and L235E andone Fc-region polypeptide comprises the mutation T366W, and the otherFc-region polypeptide comprises the mutations T366S, L368A and Y407V, orwherein one Fc-region polypeptide comprises the mutations T366W andY349C, and the other Fc-region polypeptide comprises the mutationsT366S, L368A, Y407V, and S354C, or wherein one Fc-region polypeptidecomprises the mutations T366W and S354C, and the other Fc-regionpolypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer, R. L. et al., J. Immunol. 117 (1976)587-593, and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434), aredescribed in US 2005/0014934. Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).See also Duncan, A. R. and Winter, G., Nature 322 (1988) 738-740; U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of Fc domains or cellactivating bispecific antigen binding molecules comprising an Fc domainfor Fc receptors may be evaluated using cell lines known to expressparticular Fc receptors, such as human NK cells expressing FcγIIIareceptor. Effector function of an Fc domain, or bispecific antibodies ofthe invention comprising an Fc domain, can be measured by methods knownin the art. A suitable assay for measuring ADCC is described herein.Other examples of in vitro assays to assess ADCC activity of a moleculeof interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al.Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., ProcNatl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337;Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.); and CytoTox 96© non-radioactivecytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g. in a animalmodel such as that disclosed in Clynes et al., Proc Natl Acad Sci USA95, 652-656 (1998).

Fc Domain Modifications Promoting Heterodimerization

The bispecific antigen binding molecules of the invention comprisedifferent antigen-binding sites, fused to one or the other of the twosubunits of the Fc domain, thus the two subunits of the Fc domain may becomprised in two non-identical polypeptide chains. Recombinantco-expression of these polypeptides and subsequent dimerization leads toseveral possible combinations of the two polypeptides. To improve theyield and purity of the bispecific antibodies of the invention inrecombinant production, it will thus be advantageous to introduce in theFc domain of the bispecific antigen binding molecules of the invention amodification promoting the association of the desired polypeptides.

Accordingly, in particular aspects the invention relates to a bispecificantibody comprising a first antigen-binding site that specifically bindsto PD1 and a second antigen-binding site that specifically binds toTIM3, wherein the Fc domain comprises a modification promoting theassociation of the first and second subunit of the Fc domain. The siteof most extensive protein-protein interaction between the two subunitsof a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, inone aspect said modification is in the CH3 domain of the Fc domain.

In a specific aspect said modification is a so-called “knob-into-hole”modification, comprising a “knob” modification in one of the twosubunits of the Fc domain and a “hole” modification in the other one ofthe two subunits of the Fc domain. Thus, the invention relates to abispecific antibody comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM3, wherein the first subunit of the Fc domaincomprises knobs and the second subunit of the Fc domain comprises holesaccording to the knobs into holes method. In a particular aspect, thefirst subunit of the Fc domain comprises the amino acid substitutionsS354C and T366W (EU numbering) and the second subunit of the Fc domaincomprises the amino acid substitutions Y349C, T366S and Y407V (numberingaccording to Kabat EU index).

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in one aspect, in the CH3 domain of the first subunit ofthe Fc domain of the bispecific antigen binding molecules of theinvention an amino acid residue is replaced with an amino acid residuehaving a larger side chain volume, thereby generating a protuberancewithin the CH3 domain of the first subunit which is positionable in acavity within the CH3 domain of the second subunit, and in the CH3domain of the second subunit of the Fc domain an amino acid residue isreplaced with an amino acid residue having a smaller side chain volume,thereby generating a cavity within the CH3 domain of the second subunitwithin which the protuberance within the CH3 domain of the first subunitis positionable. The protuberance and cavity can be made by altering thenucleic acid encoding the polypeptides, e.g. by site-specificmutagenesis, or by peptide synthesis. In a specific aspect, in the CH3domain of the first subunit of the Fc domain the threonine residue atposition 366 is replaced with a tryptophan residue (T366W), and in theCH3 domain of the second subunit of the Fc domain the tyrosine residueat position 407 is replaced with a valine residue (Y407V). In oneaspect, in the second subunit of the Fc domain additionally thethreonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A).

In yet a further aspect, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fc domainadditionally the tyrosine residue at position 349 is replaced by acysteine residue (Y349C). Introduction of these two cysteine residuesresults in formation of a disulfide bridge between the two subunits ofthe Fc domain, further stabilizing the dimer (Carter (2001), J ImmunolMethods 248, 7-15). In a particular aspect, the first subunit of the Fcdomain comprises the amino acid substitutions S354C and T366W (EUnumbering) and the second subunit of the Fc domain comprises the aminoacid substitutions Y349C, T366S and Y407V (numbering according to KabatEU index).

But also other knobs-in-holes technologies as described by EP 1 870 459,can be used alternatively or additionally. In one embodiment themultispecific antibody comprises the mutations R409D and K370E in theCH3 domain of the “knobs chain” and the mutations D399K and E357K in theCH3 domain of the “hole-chain” (numbering according to Kabat EU index).

In one aspect, the bispecific antibody comprises a T366W mutation in theCH3 domain of the “knobs chain” and the mutations T366S, L368A and Y407Vin the CH3 domain of the “hole chain” and additionally the mutationsR409D and K370E in the CH3 domain of the “knobs chain” and the mutationsD399K and E357K in the CH3 domain of the “hole chain” (numberingaccording to the Kabat EU index).

In one aspect, the bispecific antibody comprises the mutations Y349C andT366W in one of the two CH3 domains and the mutations S354C, T366S,L368A and Y407V in the other of the two CH3 domains, or themultispecific antibody comprises the mutations Y349C and T366W in one ofthe two CH3 domains and the mutations S354C, T366S, L368A and Y407V inthe other of the two CH3 domains and additionally the mutations R409Dand K370E in the CH3 domain of the “knobs chain” and the mutations D399Kand E357K in the CH3 domain of the “hole chain” (numbering according tothe Kabat EU index).

In an alternative aspect, a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

Apart from the “knob-into-hole technology” other techniques formodifying the CH3 domains of the heavy chains of a multispecificantibody to enforce heterodimerization are known in the art. Thesetechnologies, especially the ones described in WO 96/27011, WO98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004,WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO2013/157954 and WO 2013/096291 are contemplated herein as alternativesto the “knob-into-hole technology” in combination with a bispecificantibody.

In one aspect, in the bispecific antibody the approach described in EP1870459 is used to support heterodimerization of the first heavy chainand the second heavy chain of the multispecific antibody. This approachis based on the introduction of charged amino acids with oppositecharges at specific amino acid positions in the CH3/CH3-domain-interfacebetween both, the first and the second heavy chain.

Accordingly, in this aspect in the tertiary structure of themultispecific antibody the CH3 domain of the first heavy chain and theCH3 domain of the second heavy chain form an interface that is locatedbetween the respective antibody CH3 domains, wherein the respectiveamino acid sequences of the CH3 domain of the first heavy chain and theamino acid sequence of the CH3 domain of the second heavy chain eachcomprise a set of amino acids that is located within said interface inthe tertiary structure of the antibody, wherein from the set of aminoacids that is located in the interface in the CH3 domain of one heavychain a first amino acid is substituted by a positively charged aminoacid and from the set of amino acids that is located in the interface inthe CH3 domain of the other heavy chain a second amino acid issubstituted by a negatively charged amino acid. The bispecific antibodyaccording to this aspect is herein also referred to as“CH3(+/−)-engineered bispecific antibody” (wherein the abbreviation“+/−” stands for the oppositely charged amino acids that were introducedin the respective CH3 domains).

In one aspect, in the CH3(+/−)-engineered bispecific antibody thepositively charged amino acid is selected from K, R and H, and thenegatively charged amino acid is selected from E or D.

In one aspect, in the CH3(+/−)-engineered bispecific antibody thepositively charged amino acid is selected from K and R, and thenegatively charged amino acid is selected from E or D.

In one aspect, in the CH3(+/−)-engineered bispecific antibody thepositively charged amino acid is K, and the negatively charged aminoacid is E.

In one aspect, in the CH3(+/−)-engineered bispecific antibody in the CH3domain of one heavy chain the amino acid R at position 409 issubstituted by D and the amino acid K at position is substituted by E,and in the CH3 domain of the other heavy chain the amino acid D atposition 399 is substituted by K and the amino acid E at position 357 issubstituted by K (numbering according to Kabat EU index).

In one aspect, the approach described in WO 2013/157953 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one embodiment in the CH3 domainof one heavy chain the amino acid T at position 366 is substituted by K,and in the CH3 domain of the other heavy chain the amino acid L atposition 351 is substituted by D (numbering according to Kabat EUindex). In another embodiment in the CH3 domain of one heavy chain theamino acid T at position 366 is substituted by K and the amino acid L atposition 351 is substituted by K, and in the CH3 domain of the otherheavy chain the amino acid L at position 351 is substituted by D(numbering according to Kabat EU index).

In another aspect, in the CH3 domain of one heavy chain the amino acid Tat position 366 is substituted by K and the amino acid L at position 351is substituted by K, and in the CH3 domain of the other heavy chain theamino acid L at position 351 is substituted by D (numbering according toKabat EU index). Additionally at least one of the followingsubstitutions is comprised in the CH3 domain of the other heavy chain:the amino acid Y at position 349 is substituted by E, the amino acid Yat position 349 is substituted by D and the amino acid L at position 368is substituted by E (numbering according to Kabat EU index). In oneembodiment the amino acid L at position 368 is substituted by E(numbering according to Kabat EU index).

In one aspect, the approach described in WO 2012/058768 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one aspect, in the CH3 domain ofone heavy chain the amino acid L at position 351 is substituted by Y andthe amino acid Y at position 407 is substituted by A, and in the CH3domain of the other heavy chain the amino acid T at position 366 issubstituted by A and the amino acid K at position 409 is substituted byF (numbering according to Kabat EU index). In another embodiment, inaddition to the aforementioned substitutions, in the CH3 domain of theother heavy chain at least one of the amino acids at positions 411(originally T), 399 (originally D), 400 (originally S), 405 (originallyF), 390 (originally N) and 392 (originally K) is substituted (numberingaccording to Kabat EU index). Preferred substitutions are:

-   -   substituting the amino acid T at position 411 by an amino acid        selected from N, R, Q, K, D, E and W (numbering according to        Kabat EU index),    -   substituting the amino acid D at position 399 by an amino acid        selected from R, W, Y, and K (numbering according to Kabat EU        index),    -   substituting the amino acid S at position 400 by an amino acid        selected from E, D, R and K (numbering according to Kabat EU        index),    -   substituting the amino acid F at position 405 by an amino acid        selected from I, M, T, S, V and W (numbering according to Kabat        EU index;    -   substituting the amino acid N at position 390 by an amino acid        selected from R, K and D (numbering according to Kabat EU index;        and    -   substituting the amino acid K at position 392 by an amino acid        selected from V, M, R, L, F and E (numbering according to Kabat        EU index).

In another aspect, the bispecific antibody is engineered according to WO2012/058768), i.e. in the CH3 domain of one heavy chain the amino acid Lat position 351 is substituted by Y and the amino acid Y at position 407is substituted by A, and in the CH3 domain of the other heavy chain theamino acid T at position 366 is substituted by V and the amino acid K atposition 409 is substituted by F (numbering according to Kabat EUindex). In another embodiment of the multispecific antibody, in the CH3domain of one heavy chain the amino acid Y at position 407 issubstituted by A, and in the CH3 domain of the other heavy chain theamino acid T at position 366 is substituted by A and the amino acid K atposition 409 is substituted by F (numbering according to Kabat EUindex). In the last aforementioned embodiment, in the CH3 domain of theother heavy chain the amino acid K at position 392 is substituted by E,the amino acid T at position 411 is substituted by E, the amino acid Dat position 399 is substituted by R and the amino acid S at position 400is substituted by R (numbering according to Kabat EU index).

In one aspect, the approach described in WO 2011/143545 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one aspect, amino acidmodifications in the CH3 domains of both heavy chains are introduced atpositions 368 and/or 409 (numbering according to Kabat EU index).

In one aspect, the approach described in WO 2011/090762 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the bispecific antibody. WO 2011/090762 relates to amino acidmodifications according to the “knob-into-hole” (KiH) technology. In oneembodiment in the CH3 domain of one heavy chain the amino acid T atposition 366 is substituted by W, and in the CH3 domain of the otherheavy chain the amino acid Y at position 407 is substituted by A(numbering according to Kabat EU index). In another embodiment in theCH3 domain of one heavy chain the amino acid T at position 366 issubstituted by Y, and in the CH3 domain of the other heavy chain theamino acid Y at position 407 is substituted by T (numbering according toKabat EU index).

In one aspect, the approach described in WO 2009/089004 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the bispecific antibody. In one embodiment in the CH3 domain ofone heavy chain the amino acid K or N at position 392 is substituted bya negatively charged amino acid (in one embodiment by E or D, in onepreferred embodiment by D), and in the CH3 domain of the other heavychain the amino acid D at position 399 the amino acid E or D at position356 or the amino acid E at position 357 is substituted by a positivelycharged amino acid (in one embodiment K or R, in one preferredembodiment by K, in one preferred embodiment the amino acids atpositions 399 or 356 are substituted by K) (numbering according to KabatEU index). In one further embodiment, in addition to the aforementionedsubstitutions, in the CH3 domain of the one heavy chain the amino acid Kor R at position 409 is substituted by a negatively charged amino acid(in one embodiment by E or D, in one preferred embodiment by D)(numbering according to Kabat EU index). In one even further aspect, inaddition to or alternatively to the aforementioned substitutions, in theCH3 domain of the one heavy chain the amino acid K at position 439and/or the amino acid K at position 370 is substituted independentlyfrom each other by a negatively charged amino acid (in one embodiment byE or D, in one preferred embodiment by D) (numbering according to KabatEU index).

In one aspect, the approach described in WO 2007/147901 is used tosupport heterodimerization of the first heavy chain and the second heavychain of the multispecific antibody. In one embodiment in the CH3 domainof one heavy chain the amino acid K at position 253 is substituted by E,the amino acid D at position 282 is substituted by K and the amino acidK at position 322 is substituted by D, and in the CH3 domain of theother heavy chain the amino acid D at position 239 is substituted by K,the amino acid E at position 240 is substituted by K and the amino acidK at position 292 is substituted by D (numbering according to Kabat EUindex).

The C-terminus of the heavy chain of the bispecific antibody as reportedherein can be a complete C-terminus ending with the amino acid residuesPGK. The C-terminus of the heavy chain can be a shortened C-terminus inwhich one or two of the C terminal amino acid residues have beenremoved. In one preferred aspect, the C-terminus of the heavy chain is ashortened C-terminus ending PG.

In one aspect of all aspects as reported herein, a bispecific antibodycomprising a heavy chain including a C-terminal CH3 domain as specifiedherein, comprises the C-terminal glycine-lysine dipeptide (G446 andK447, numbering according to Kabat EU index). In one embodiment of allaspects as reported herein, a bispecific antibody comprising a heavychain including a C-terminal CH3 domain, as specified herein, comprisesa C-terminal glycine residue (G446, numbering according to Kabat EUindex).

Modifications in the Fab Domains

In one aspect, the invention relates to a bispecific antibody comprisinga first Fab fragment that specifically binds to PD1 and a second Fabfragment that specifically binds to TIM3, wherein in one of the Fabfragments either the variable domains VH and VL or the constant domainsCH1 and CL are exchanged. The bispecific antibodies are preparedaccording to the Crossmab technology.

Multispecific antibodies with a domain replacement/exchange in onebinding arm (CrossMabVH-VL or CrossMabCH-CL) are described in detail inWO2009/080252 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191. Theyclearly reduce the byproducts caused by the mismatch of a light chainagainst a first antigen with the wrong heavy chain against the secondantigen (compared to approaches without such domain exchange).

In a particular aspect, the invention relates to a bispecific antibodycomprising a first Fab fragment that specifically binds to PD1 and asecond Fab fragment that specifically binds to TIM3, wherein in one ofthe Fab fragments the variable domains VL and VH are replaced by eachother so that the VH domain is part of the light chain and the VL domainis part of the heavy chain. In a particular aspect, the bispecificantibody is one, wherein in the first Fab fragment comprising theantigen-binding site that specifically binds to PD1 the variable domainsVL and VH are replaced by each other.

In another aspect, and to further improve correct pairing, thebispecific antibody comprising a first Fab fragment that specificallybinds to PD1 and a second Fab fragment that specifically binds to TIM3,can contain different charged amino acid substitutions (so-called“charged residues”). These modifications are introduced in the crossedor non-crossed CH1 and CL domains.

In a particular aspect, the invention is concerned with a bispecificantibody comprising a first Fab fragment that specifically binds to PD1and a second Fab fragment that specifically binds to TIM3, wherein inone of the Fab fragments in the constant domain CL the amino acid atposition 124 is substituted independently by lysine (K), arginine (R) orhistidine (H) (numbering according to Kabat EU Index), and in theconstant domain CH1 the amino acids at positions 147 and 213 aresubstituted independently by glutamic acid (E) or aspartic acid (D)(numbering according to Kabat EU index). In a particular aspect, thebispecific antibody is one, wherein in the second Fab fragmentcomprising the antigen-binding site that specifically binds to TIM3 theconstant domain CL the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat EU Index), and in the constant domain CH1 the aminoacids at positions 147 and 213 are substituted independently by glutamicacid (E) or aspartic acid (D) (numbering according to Kabat EU index).

In a particular aspect, the invention relates to a bispecific antibodycomprising a first Fab fragment that specifically binds to PD1 and asecond Fab fragment that specifically binds to TIM3, wherein in one ofCL domains the amino acid at position 123 (EU numbering) has beenreplaced by arginine (R) and the amino acid at position 124 (EUnumbering) has been substituted by lysine (K) and wherein in one of theCH1 domains the amino acids at position 147 (EU numbering) and atposition 213 (EU numbering) have been substituted by glutamic acid (E).In a particular aspect, the bispecific antibody is one, wherein in thesecond Fab fragment comprising the antigen-binding site thatspecifically binds to TIM3 the amino acid at position 123 (EU numbering)has been replaced by arginine (R) and the amino acid at position 124 (EUnumbering) has been substituted by lysine (K) and wherein in one of theCH1 domains the amino acids at position 147 (EU numbering) and atposition 213 (EU numbering) have been substituted by glutamic acid (E).

In a further aspect, the bispecific antibody is a bivalent antibodycomprising

a) a first light chain and a first heavy chain of an antibodyspecifically binding to a first antigen, and

b) a second light chain and a second heavy chain of an antibodyspecifically binding to a second antigen, wherein the variable domainsVL and VH of the second light chain and the second heavy chain arereplaced by each other.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain under a) are isolated chains.

In the antibody under b) within the light chain the variable light chaindomain VL is replaced by the variable heavy chain domain VH of saidantibody, and within the heavy chain the variable heavy chain domain VHis replaced by the variable light chain domain VL of said antibody.

In one aspect, (i) in the constant domain CL of the first light chainunder a) the amino acid at position 124 (numbering according to Kabat)is substituted by a positively charged amino acid, and wherein in theconstant domain CH1 of the first heavy chain under a) the amino acid atposition 147 or the amino acid at position 213 (numbering according toKabat EU index) is substituted by a negatively charged amino acid, or(ii) in the constant domain CL of the second light chain under b) theamino acid at position 124 (numbering according to Kabat) is substitutedby a positively charged amino acid, and wherein in the constant domainCH1 of the second heavy chain under b) the amino acid at position 147 orthe amino acid at position 213 (numbering according to Kabat EU index)is substituted by a negatively charged amino acid.

In another aspect, (i) in the constant domain CL of the first lightchain under a) the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat) (in one preferred embodiment independently by lysine(K) or arginine (R)), and wherein in the constant domain CH1 of thefirst heavy chain under a) the amino acid at position 147 or the aminoacid at position 213 is substituted independently by glutamic acid (E)or aspartic acid (D) (numbering according to Kabat EU index), or (ii) inthe constant domain CL of the second light chain under b) the amino acidat position 124 is substituted independently by lysine (K), arginine (R)or histidine (H) (numbering according to Kabat) (in one preferredembodiment independently by lysine (K) or arginine (R)), and wherein inthe constant domain CH1 of the second heavy chain under b) the aminoacid at position 147 or the amino acid at position 213 is substitutedindependently by glutamic acid (E) or aspartic acid (D) (numberingaccording to Kabat EU index).

In one aspect, in the constant domain CL of the second heavy chain theamino acids at position 124 and 123 are substituted by K (numberingaccording to Kabat EU index).

In one aspect, in the constant domain CL of the second heavy chain theamino acid at position 123 is substituted by R and the amino acid asposition 124 is substituted by K (numbering according to Kabat EUindex).

In one aspect, in the constant domain CH1 of the second light chain theamino acids at position 147 and 213 are substituted by E (numberingaccording to EU index of Kabat).

In one aspect, in the constant domain CL of the first light chain theamino acids at position 124 and 123 are substituted by K, and in theconstant domain CH1 of the first heavy chain the amino acids at position147 and 213 are substituted by E (numbering according to Kabat EUindex).

In one aspect, in the constant domain CL of the first light chain theamino acid at position 123 is substituted by R and the amino acid atposition 124 is substituted by K, and in the constant domain CH1 of thefirst heavy chain the amino acids at position 147 and 213 are bothsubstituted by E (numbering according to Kabat EU index).

In one aspect, in the constant domain CL of the second heavy chain theamino acids at position 124 and 123 are substituted by K, and wherein inthe constant domain CH1 of the second light chain the amino acids atposition 147 and 213 are substituted by E, and in the variable domain VLof the first light chain the amino acid at position 38 is substituted byK, in the variable domain VH of the first heavy chain the amino acid atposition 39 is substituted by E, in the variable domain VL of the secondheavy chain the amino acid at position 38 is substituted by K, and inthe variable domain VH of the second light chain the amino acid atposition 39 is substituted by E (numbering according to Kabat EU index).

In one aspect, the bispecific antibody is a bivalent antibody comprising

a) a first light chain and a first heavy chain of an antibodyspecifically binding to a first antigen, and

b) a second light chain and a second heavy chain of an antibodyspecifically binding to a second antigen, wherein the variable domainsVL and VH of the second light chain and the second heavy chain arereplaced by each other, and wherein the constant domains CL and CH1 ofthe second light chain and the second heavy chain are replaced by eachother.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain und a) are isolated chains.In the antibody under b) within the light chain the variable light chaindomain VL is replaced by the variable heavy chain domain VH of saidantibody, and the constant light chain domain CL is replaced by theconstant heavy chain domain CH1 of said antibody; and within the heavychain the variable heavy chain domain VH is replaced by the variablelight chain domain VL of said antibody, and the constant heavy chaindomain CH1 is replaced by the constant light chain domain CL of saidantibody.

In one aspect, the bispecific antibody is a bivalent antibody comprising

a) a first light chain and a first heavy chain of an antibodyspecifically binding to a first antigen, and

b) a second light chain and a second heavy chain of an antibodyspecifically binding to a second antigen, wherein the constant domainsCL and CH1 of the second light chain and the second heavy chain arereplaced by each other.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain under a) are isolated chains.In the antibody under b) within the light chain the constant light chaindomain CL is replaced by the constant heavy chain domain CH1 of saidantibody; and within the heavy chain the constant heavy chain domain CH1is replaced by the constant light chain domain CL of said antibody.

In one aspect, the multispecific antibody is a multispecific antibodycomprising

a) a full length antibody specifically binding to a first antigen andconsisting of two antibody heavy chains and two antibody light chains,and

b) one, two, three or four single chain Fab fragments specificallybinding to one to four further antigens (i.e. a second and/or thirdand/or fourth and/or fifth antigen, preferably specifically binding toone further antigen, i.e. a second antigen), wherein said single chainFab fragments under b) are fused to said full length antibody under a)via a peptidic linker at the C- or N-terminus of the heavy or lightchain of said full length antibody.

In one aspect, one or two identical single chain Fab fragments bindingto a second antigen are fused to the full length antibody via a peptidiclinker at the C terminus of the heavy or light chains of said fulllength antibody.

In one aspect, one or two identical single chain Fab fragments bindingto a second antigen are fused to the full length antibody via a peptidiclinker at the C terminus of the heavy chains of said full lengthantibody.

In one aspect, one or two identical single chain Fab fragments bindingto a second antigen are fused to the full length antibody via a peptidiclinker at the C terminus of the light chains of said full lengthantibody.

In one aspect, two identical single chain Fab fragments binding to asecond antigen are fused to the full length antibody via a peptidiclinker at the C-terminus of each heavy or light chain of said fulllength antibody.

In one aspect, two identical single chain Fab fragments binding to asecond antigen are fused to the full length antibody via a peptidiclinker at the C-terminus of each heavy chain of said full lengthantibody.

In one aspect, two identical single chain Fab fragments binding to asecond antigen are fused to the full length antibody via a peptidiclinker at the C-terminus of each light chain of said full lengthantibody.

In one aspect, the bispecific antibody is a trivalent antibodycomprising

a) a full length antibody specifically binding to a first antigen andconsisting of two antibody heavy chains and two antibody light chains,

b) a first polypeptide consisting of

-   -   ba) an antibody heavy chain variable domain (VH), or    -   bb) an antibody heavy chain variable domain (VH) and an antibody        constant domain 1 (CH1),

wherein said first polypeptide is fused with the N-terminus of its VHdomain via a peptidic linker to the C-terminus of one of the two heavychains of said full length antibody,

c) a second polypeptide consisting of

-   -   ca) an antibody light chain variable domain (VL), or    -   cb) an antibody light chain variable domain (VL) and an antibody        light chain constant domain (CL),

wherein said second polypeptide is fused with the N-terminus of the VLdomain via a peptidic linker to the C-terminus of the other of the twoheavy chains of said full length antibody, and

wherein the antibody heavy chain variable domain (VH) of the firstpolypeptide and the antibody light chain variable domain (VL) of thesecond polypeptide together form an antigen-binding site specificallybinding to a second antigen.

In one aspect, the antibody heavy chain variable domain (VH) of thepolypeptide under b) and the antibody light chain variable domain (VL)of the polypeptide under c) are linked and stabilized via an interchaindisulfide bridge by introduction of a disulfide bond between thefollowing positions:

(i) heavy chain variable domain position 44 to light chain variabledomain position 100, or

(ii) heavy chain variable domain position 105 to light chain variabledomain position 43, or

(iii) heavy chain variable domain position 101 to light chain variabledomain position 100 (numbering always according to Kabat EU index).

Techniques to introduce unnatural disulfide bridges for stabilizationare described e.g. in WO 94/029350, Rajagopal, V., et al., Prot. Eng.(1997) 1453-1459; Kobayashi, H., et al., Nucl. Med. Biol. 25 (1998)387-393; and Schmidt, M., et al., Oncogene 18 (1999) 1711-1721. In oneembodiment the optional disulfide bond between the variable domains ofthe polypeptides under b) and c) is between heavy chain variable domainposition 44 and light chain variable domain position 100. In oneembodiment the optional disulfide bond between the variable domains ofthe polypeptides under b) and c) is between heavy chain variable domainposition 105 and light chain variable domain position 43 (numberingalways according to Kabat). In one embodiment a trivalent, bispecificantibody without said optional disulfide stabilization between thevariable domains VH and VL of the single chain Fab fragments ispreferred.

In one aspect, the bispecific antibody is a trispecific or tetraspecificantibody, comprising

a) a first light chain and a first heavy chain of a full length antibodywhich specifically binds to a first antigen, and

b) a second (modified) light chain and a second (modified) heavy chainof a full length antibody which specifically binds to a second antigen,wherein the variable domains VL and VH are replaced by each other,and/or wherein the constant domains CL and CH1 are replaced by eachother, and

c) wherein one to four antigen binding peptides which specifically bindto one or two further antigens (i.e. to a third and/or fourth antigen)are fused via a peptidic linker to the C- or N-terminus of the lightchains or heavy chains of a) and/or b).

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain und a) are isolated chains.

In one aspect, the trispecific or tetraspecific antibody comprises underc) one or two antigen binding peptides which specifically bind to one ortwo further antigens.

In one aspect, the antigen binding peptides are selected from the groupof a scFv fragment and a scFab fragment.

In one aspect, the antigen binding peptides are scFv fragments.

In one aspect, the antigen binding peptides are scFab fragments.

In one aspect, the antigen binding peptides are fused to the C-terminusof the heavy chains of a) and/or b).

In one aspect, the trispecific or tetraspecific antibody comprises underc) one or two antigen binding peptides which specifically bind to onefurther antigen.

In one aspect, the trispecific or tetraspecific antibody comprises underc) two identical antigen binding peptides which specifically bind to athird antigen. In one preferred embodiment such two identical antigenbinding peptides are fused both via the same peptidic linker to theC-terminus of the heavy chains of a) and b). In one preferred embodimentthe two identical antigen binding peptides are either a scFv fragment ora scFab fragment.

In one aspect, the trispecific or tetraspecific antibody comprises underc) two antigen binding peptides which specifically bind to a third and afourth antigen. In one embodiment said two antigen binding peptides arefused both via the same peptide connector to the C-terminus of the heavychains of a) and b). In one preferred embodiment said two antigenbinding peptides are either a scFv fragment or a scFab fragment.

In one aspect, the bispecific antibody is a bispecific, tetravalentantibody comprising

a) two light chains and two heavy chains of an antibody, whichspecifically bind to a first antigen (and comprise two Fab fragments),

b) two additional Fab fragments of an antibody, which specifically bindto a second antigen, wherein said additional Fab fragments are fusedboth via a peptidic linker either to the C- or N-termini of the heavychains of a), and

wherein in the Fab fragments the following modifications were performed

(i) in both Fab fragments of a), or in both Fab fragments of b), thevariable domains VL and VH are replaced by each other, and/or theconstant domains CL and CH1 are replaced by each other, or

(ii) in both Fab fragments of a) the variable domains VL and VH arereplaced by each other, and the constant domains CL and CH1 are replacedby each other, and in both Fab fragments of b) the variable domains VLand VH are replaced by each other, or the constant domains CL and CH1are replaced by each other, or

(iii) in both Fab fragments of a) the variable domains VL and VH arereplaced by each other, or the constant domains CL and CH1 are replacedby each other, and in both Fab fragments of b) the variable domains VLand VH are replaced by each other, and the constant domains CL and CH1are replaced by each other, or

(iv) in both Fab fragments of a) the variable domains VL and VH arereplaced by each other, and in both Fab fragments of b) the constantdomains CL and CH1 are replaced by each other, or

(v) in both Fab fragments of a) the constant domains CL and CH1 arereplaced by each other, and in both Fab fragments of b) the variabledomains VL and VH are replaced by each other.

In one aspect, said additional Fab fragments are fused both via apeptidic linker either to the C-termini of the heavy chains of a), or tothe N-termini of the heavy chains of a).

In one aspect, said additional Fab fragments are fused both via apeptidic linker either to the C-termini of the heavy chains of a).

In one aspect, said additional Fab fragments are fused both via apeptide connector to the N-termini of the heavy chains of a).

In one aspect, in the Fab fragments the following modifications areperformed: in both Fab fragments of a), or in both Fab fragments of b),the variable domains VL and VH are replaced by each other, and/or theconstant domains CL and CH1 are replaced by each other.

In one aspect, the bispecific antibody is a tetravalent antibodycomprising:

a) a (modified) heavy chain of a first antibody, which specificallybinds to a first antigen and comprises a first VH-CH1 domain pair,wherein to the C terminus of said heavy chain the N-terminus of a secondVH-CH1 domain pair of said first antibody is fused via a peptidiclinker,

b) two light chains of said first antibody of a),

c) a (modified) heavy chain of a second antibody, which specificallybinds to a second antigen and comprises a first VH-CL domain pair,wherein to the C-terminus of said heavy chain the N-terminus of a secondVH-CL domain pair of said second antibody is fused via a peptidiclinker, and

d) two (modified) light chains of said second antibody of c), eachcomprising a CL-CH1 domain pair.

In one aspect, the bispecific antibody comprises

a) the heavy chain and the light chain of a first full length antibodythat specifically binds to a first antigen, and

b) the heavy chain and the light chain of a second full length antibodythat specifically binds to a second antigen, wherein the N-terminus ofthe heavy chain is connected to the C-terminus of the light chain via apeptidic linker.

The antibody under a) does not contain a modification as reported underb) and the heavy chain and the light chain are isolated chains.

In one aspect, the bispecific antibody comprises

a) a full length antibody specifically binding to a first antigen andconsisting of two antibody heavy chains and two antibody light chains,and

b) an Fv fragment specifically binding to a second antigen comprising aVH2 domain and a VL2 domain, wherein both domains are connected to eachother via a disulfide bridge,

wherein only either the VH2 domain or the VL2 domain is fused via apeptidic linker to the heavy or light chain of the full length antibodyspecifically binding to a first antigen.

In the bispecific antibody the heavy chains and the light chains undera) are isolated chains.

In one aspect, the other of the VH2 domain or the VL2 domain is notfused via a peptide linker to the heavy or light chain of the fulllength antibody specifically binding to a first antigen.

In all aspects as reported herein the first light chain comprises a VLdomain and a CL domain and the first heavy chain comprises a VH domain,a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

In one aspect, the bispecific antibody is a trivalent antibodycomprising

a) two Fab fragments that specifically binds to a first antigen,

b) one CrossFab fragment that specifically binds to a second antigen inwhich the CH1 and the CL domain are exchanged for each other,

c) one Fc-region comprising a first Fc-region heavy chain and a secondFc region heavy chain,

wherein the C-terminus of CH1 domains of the two Fab fragments areconnected to the N-terminus of the heavy chain Fc-region polypeptides,and wherein the C-terminus of the CL domain of the CrossFab fragment isconnected to the N-terminus of the VH domain of one of the Fabfragments.

In one aspect, the bispecific antibody is a trivalent antibodycomprising

a) two Fab fragments that specifically binds to a first antigen,

b) one CrossFab fragment that specifically binds to a second antigen inwhich the CH1 and the CL domain are exchanged for each other,

c) one Fc-region comprising a first Fc-region heavy chain and a secondFc region heavy chain,

wherein the C-terminus of CH1 domain of the first Fab fragment isconnected to the N-terminus of one of the heavy chain Fc-regionpolypeptides and the C-terminus of the CL-domain of the CrossFabfragment is connected to the N-terminus of the other heavy chainFc-region polypeptide, and wherein the C-terminus of the CH1 domain ofthe second Fab fragment is connected to the N-terminus of the VH domainof the first Fab fragment or to the N-terminus of the VH domain of theCrossFab fragment.

In one aspect, the bispecific antibody comprises

a) a full length antibody specifically binding to a first antigen andconsisting of two antibody heavy chains and two antibody light chains,and

b) a Fab fragment specifically binding to a second antigen comprising aVH2 domain and a VL2 domain comprising a heavy chain fragment and alight chain fragment, wherein within the light chain fragment thevariable light chain domain VL2 is replaced by the variable heavy chaindomain VH2 of said antibody, and within the heavy chain fragment thevariable heavy chain domain VH2 is replaced by the variable light chaindomain VL2 of said antibody

wherein the heavy chain Fab fragment is inserted between the CH1 domainof one of the heavy chains of the full length antibody and therespective Fc-region of the full length antibody, and the N-terminus ofthe light chain Fab fragment is conjugated to the C-terminus of thelight chain of the full length antibody that is paired with the heavychain of the full length antibody into which the heavy chain Fabfragment has been inserted.

In one aspect, the bispecific antibody comprises

a) a full length antibody specifically binding to a first antigen andconsisting of two antibody heavy chains and two antibody light chains,and

b) a Fab fragment specifically binding to a second antigen comprising aVH2 domain and a VL2 domain comprising a heavy chain fragment and alight chain fragment, wherein within the light chain fragment thevariable light chain domain VL2 is replaced by the variable heavy chaindomain VH2 of said antibody, and within the heavy chain fragment thevariable heavy chain domain VH2 is replaced by the variable light chaindomain VL2 of said antibody and wherein the C-terminus of the heavychain fragment of the Fab fragment is conjugated to the N-terminus ofone of the heavy chains of the full length antibody and the C-terminusof the light chain fragment of the Fab fragment is conjugated to theN-terminus of the light chain of the full length antibody that pairswith the heavy chain of the full length antibody to which the heavychain fragment of the Fab fragment is conjugated.

Polynucleotides

The invention further provides isolated polynucleotides encoding abispecific antibody as described herein or a fragment thereof.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

B. Recombinant Methods

The bispecific antibodies provided herein may be produced usingrecombinant methods and compositions, e.g., as described in U.S. Pat.No. 4,816,567. In one aspect, isolated nucleic acid encoding anbispecific antibody described herein is provided. Such nucleic acid mayencode an amino acid sequence comprising the VL and/or an amino acidsequence comprising the VH of the antigen-binding sites thatspecifically bind to PD1 and TIM-3, respectively (e.g., in the lightand/or heavy chains of the antibody). In a further aspect, one or morevectors (e.g., expression vectors) comprising such nucleic acid areprovided. In a further aspect, a host cell comprising such nucleic acidis provided. In one such aspect, a host cell comprises (e.g., has beentransformed with): (1) a vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and an aminoacid sequence comprising the VH of the antibody, or (2) a first vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and a second vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VH of the antibody.In one aspect, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary(CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one aspect, amethod of making a bispecific antibody is provided, wherein the methodcomprises culturing a host cell comprising a nucleic acid encoding theantibody, as provided above, under conditions suitable for expression ofthe antibody, and optionally recovering the antibody from the host cell(or host cell culture medium).

For recombinant production of the bispecific antibodies comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM-3 as describedherein, nucleic acid encoding the bispecific antibodies, e.g., asdescribed above, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Such nucleic acid maybe readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, K. A., In:Methods in Molecular Biology, Vol. 248, Lo, B. K. C. (ed.), HumanaPress, Totowa, N.J. (2003), pp. 245-254, describing expression ofantibody fragments in E. coli.) After expression, the antibody may beisolated from the bacterial cell paste in a soluble fraction and can befurther purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, T. U., Nat. Biotech. 22 (2004) 1409-1414; andLi, H. et al., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibodies arealso derived from multicellular organisms (invertebrates andvertebrates). Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains have been identified which may beused in conjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham, F. L. et al., J. Gen Virol. 36(1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4cells as described, e.g., in Mather, J. P., Biol. Reprod. 23 (1980)243-252); monkey kidney cells (CV1); African green monkey kidney cells(VERO-76); human cervical carcinoma cells (HELA); canine kidney cells(MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); humanliver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, asdescribed, e.g., in Mather, J. P. et al., Annals N.Y. Acad. Sci. 383(1982) 44-68; MRC 5 cells; and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, including DHFR−CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980)4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki, P. and Wu, A. M., Methods in MolecularBiology, Vol. 248, Lo, B. K. C. (ed.), Humana Press, Totowa, N.J.(2004), pp. 255-268.

C. Assays

The bispecific antibodies comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM-3 provided herein may be identified, screenedfor, or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Affinity Assays

The affinity of the bispecific antigen binding molecules, antibodies andantibody fragments provided herein for the corresponding antigens can bedetermined in accordance with the methods set forth in the Examples bysurface plasmon resonance (SPR), using standard instrumentation such asa Biacore® instrument (GE Healthcare), and receptors or target proteinssuch as may be obtained by recombinant expression. A specificillustrative and exemplary embodiment for measuring binding affinity isdescribed in Examples 1b, 5 or 12. According to one aspect, K_(D) ismeasured by surface plasmon resonance using a BIACORE® T100 machine (GEHealthcare) at 25° C.

2. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc. Binding of the bispecific antibodies provided herein to thecorresponding recombinant antigen or to antigen-expressing cells may beevaluated by ELISA as described in Example 12.

In another aspect, the invention provides a cell-based TR-FRET assay todetermine the simultaneous binding of bispecific antibody formats to twodifferent receptors present on one cell. The chosen Tag-lite technologyis a combination of a classical TR-FRET (time-resolved fluorescenceresonance energy transfer) and SNAP-tag technology (e.g. New EnglandBiolabs, CISBIO), which allows antigens present on the cell surface tobe labeled with a fluorescent donor or acceptor dye. The assay isdescribed in Example 13.

In a further aspect, fresh peripheral blood mononuclear cells (PBMCs)are used in binding assays to show binding to different peripheral bloodmononuclear cells (PBMC) such as monocytes, NK cells and T cells.

In another aspect, competition assays may be used to identify anantibody that competes with a specific antibody or antigen binding sitefor binding to the target, respectively. In certain embodiments, such acompeting antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3according to the invention. Detailed exemplary methods for mapping anepitope to which an antibody binds are provided in Morris (1996)“Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66(Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized PD1 or TIM3 is incubatedin a solution comprising a first labeled antibody that binds to PD1 orTIM3 and a second unlabeled antibody that is being tested for itsability to compete with the first antibody for binding to PD1 or TIM3.The second antibody may be present in a hybridoma supernatant. As acontrol, immobilized PD1 or TIM3 is incubated in a solution comprisingthe first labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto PD1 or TIM3, excess unbound antibody is removed, and the amount oflabel associated with immobilized PD1 or TIM3 is measured. If the amountof label associated with immobilized PD1 or TIM3 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to PD1 or TIM3. See Harlow and Lane (1988) Antibodies: ALaboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.).

3. Activity Assays

In one aspect, assays are provided for identifying a bispecific antibodycomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM3 havingbiological activity. Biological activity may include, e.g., the abilityto enhance the activation and/or proliferation of different immunecells, especially T-cells, secretion of immune-modulating cytokines suchIFNγ or TNF-alpha, blocking the PD1 pathway, blocking the TIM3 pathway,killing of tumor cells. Antibodies having such biological activity invivo and/or in vitro are also provided.

In certain aspects, an antibody of the invention is tested for suchbiological activity. In one aspect, provided is an immune cell assaywhich measures the activation of lymphocytes from one individual (donorX) to lymphocytes from another individual (donor Y). The mixedlymphocyte reaction (MLR) can demonstrate the effect of blocking the PD1pathway to lymphocyte effector cells. T cells in the assay were testedfor activation and their IFN-gamma secretion in the presence or absenceof bispecific antibodies of the invention. The assay is described inmore detail in Example 16.

D. Immunoconjugates

The invention also provides immunoconjugates comprising a bispecificantibody of the invention conjugated to one or more cytotoxic agents,such as chemotherapeutic agents or drugs, growth inhibitory agents,toxins (e.g., protein toxins, enzymatically active toxins of bacterial,fungal, plant, or animal origin, or fragments thereof), or radioactiveisotopes.

In one aspect, an immunoconjugate is an antibody-drug conjugate (ADC) inwhich an antibody is conjugated to one or more drugs, including but notlimited to a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 andEuropean Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At2l1, I131, I125, Y90,Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or 1123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain aspects, any of the bispecific antibodies comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM-3 provided hereinmay be useful for detecting the presence of both PD1 and TIM3 in abiological sample. The term “detecting” as used herein encompassesquantitative or qualitative detection. In certain embodiments, abiological sample comprises a cell or tissue, such as AML stem cancercells.

In one aspect, a bispecific antibody for use in a method of diagnosis ordetection is provided. In a further aspect, a method of detecting thepresence of both PD1 and TIM3 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with a bispecific antibody as described herein under conditionspermissive for binding of the bispecific antibody to both PD1 and TIM3,and detecting whether a complex is formed between the bispecificantibody and both antigens. Such method may be an in vitro or in vivomethod. In one embodiment, the bispecific antibody is used to selectsubjects eligible for therapy with a bispecific antibody comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM-3 antibody, e.g.where PD1 and TIM3 are biomarkers for selection of patients.

In certain aspects, labeled bispecific antibodies are provided. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes 32P, 14C, 125I, 3H, and 131I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels or stable free radicals.

F. Pharmaceutical Compositions, Formulations and Routes ofAdministration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the bispecific antibodies comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM-3 provided herein,e.g., for use in any of the below therapeutic methods. In oneembodiment, a pharmaceutical composition comprises any of the bispecificantibodies provided herein and at least one pharmaceutically acceptableexcipient. In another embodiment, a pharmaceutical composition comprisesany of the bispecific antibodies provided herein and at least oneadditional therapeutic agent, e.g., as described below.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more bispecific antibodiesdissolved or dispersed in a pharmaceutically acceptable excipient. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that are generally non-toxic torecipients at the dosages and concentrations employed, i.e. do notproduce an adverse, allergic or other untoward reaction whenadministered to an animal, such as, for example, a human, asappropriate. The preparation of a pharmaceutical composition thatcontains at least one bispecific antibody and optionally an additionalactive ingredient will be known to those of skill in the art in light ofthe present disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. In particular, the compositions are lyophilized formulationsor aqueous solutions. As used herein, “pharmaceutically acceptableexcipient” includes any and all solvents, buffers, dispersion media,coatings, surfactants, antioxidants, preservatives (e.g. antibacterialagents, antifungal agents), isotonic agents, salts, stabilizers andcombinations thereof, as would be known to one of ordinary skill in theart.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the bispecific antibodies of the invention may beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiological saline buffer. The solution may contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Alternatively,the bispecific antibodies may be in powder form for constitution with asuitable vehicle, e.g., sterile pyrogen-free water, before use. Sterileinjectable solutions are prepared by incorporating the fusion proteinsof the invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated below, as required.Sterility may be readily accomplished, e.g., by filtration throughsterile filtration membranes. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The composition must be stable under theconditions of manufacture and storage, and preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Itwill be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less that 0.5 ng/mg protein.Suitable pharmaceutically acceptable excipients include, but are notlimited to: buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

Exemplary pharmaceutically acceptable excipients herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

In addition to the compositions described previously, the bispecificantibodies may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the bispecific antibodies may be formulated with suitablepolymeric or hydrophobic materials (for example as emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the bispecific antibodies of theinvention may be manufactured by means of conventional mixing,dissolving, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmaceutical compositions may be formulated in conventionalmanner using one or more physiologically acceptable carriers, diluents,excipients or auxiliaries which facilitate processing of the proteinsinto preparations that can be used pharmaceutically. Proper formulationis dependent upon the route of administration chosen.

The bispecific antibodies may be formulated into a composition in a freeacid or base, neutral or salt form. Pharmaceutically acceptable saltsare salts that substantially retain the biological activity of the freeacid or base. These include the acid addition salts, e.g. those formedwith the free amino groups of a proteinaceous composition, or which areformed with inorganic acids such as for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric ormandelic acid. Salts formed with the free carboxyl groups can also bederived from inorganic bases such as for example, sodium, potassium,ammonium, calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine. Pharmaceuticalsalts tend to be more soluble in aqueous and other protic solvents thanare the corresponding free base forms.

The composition herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the bispecific antibodies comprising a first antigen-binding sitethat specifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM-3 provided herein may be used in therapeuticmethods.

For use in therapeutic methods, bispecific antibodies comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM-3 as defined hereinbefore can be formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the agent, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners.

In one aspect, bispecific antibodies comprising a first antigen-bindingsite that specifically binds to PD1 and a second antigen-binding sitethat specifically binds to TIM-3 as defined herein for use as amedicament are provided. In further aspects, bispecific antibodiescomprising a first antigen-binding site that specifically binds to PD1and a second antigen-binding site that specifically binds to TIM-3 asdefined herein for use in treating a disease, in particular for use inthe treatment of cancer, are provided. In certain embodiments,bispecific antibodies comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM-3 for use in a method of treatment areprovided. In one embodiment, the invention provides bispecificantibodies comprising a first antigen-binding site that specificallybinds to PD1 and a second antigen-binding site that specifically bindsto TIM-3 as described herein for use in the treatment of a disease in anindividual in need thereof. In certain embodiments, the inventionprovides bispecific antibodies comprising a first antigen-binding sitethat specifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM-3 for use in a method of treating anindividual having a disease comprising administering to the individual atherapeutically effective amount of the bispecific antibody. In certainembodiments the disease to be treated is cancer. In another aspect, thedisease to be treated is a chronic viral infection like HIV, HBV, HCV,HSV1, CMV, LCMV or EBV. The subject, patient, or “individual” in need oftreatment is typically a mammal, more specifically a human.

In a further aspect, the invention provides for the use of bispecificantibodies comprising a first antigen-binding site that specificallybinds to PD1 and a second antigen-binding site that specifically bindsto TIM-3 as defined herein before in the manufacture or preparation of amedicament for the treatment of a disease in an individual in needthereof. In one embodiment, the medicament is for use in a method oftreating a disease comprising administering to an individual having thedisease a therapeutically effective amount of the medicament.

In certain aspects, the disease to be treated is a proliferativedisorder, particularly cancer. Examples of cancers include bladdercancer, brain cancer, head and neck cancer, pancreatic cancer, lungcancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer,endometrial cancer, esophageal cancer, colon cancer, colorectal cancer,rectal cancer, gastric cancer, prostate cancer, blood cancer, skincancer, squamous cell carcinoma, bone cancer, and kidney cancer. Othercell proliferation disorders that can be treated using bispecificantibodies comprising a first antigen-binding site that specificallybinds to PD1 and a second antigen-binding site that specifically bindsto TIM-3 according to the invention include, but are not limited toneoplasms located in the abdomen, bone, breast, digestive system, liver,pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous system(central and peripheral), lymphatic system, pelvic, skin, soft tissue,spleen, thoracic region, and urogenital system. Also included arepre-cancerous conditions or lesions and cancer metastases. In certainaspects, the cancer is chosen from the group consisting of renal cellcancer, skin cancer, lung cancer, colorectal cancer, breast cancer,brain cancer, head and neck cancer. In further aspects, the cancer ischosen from carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin'slymphoma), blastoma, sarcoma, and leukemia. In another aspect, thecancer is to be treated is selected from squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer,hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, leukemia and other lymphoproliferative disorders, and varioustypes of head and neck cancer.

In a further aspect, the disease to be treated is a chronic viralinfection. The term “chronic viral infection” refers to a subjectafflicted or infected with a chronic virus. Examples for chronic viralinfections are human immunodeficiency virus (HIV), hepatitis B viralinfection (HBV), hepatitis C viral infection (HCV), herpes simplex virus1 (HSV1), cytomegalovirus (CMV), lymphocytic choriomeningitis virus(LCMV) or Epstein-Barr virus (EBV).

A skilled artisan readily recognizes that in many cases the bispecificmolecule may not provide a cure but may only provide partial benefit. Insome embodiments, a physiological change having some benefit is alsoconsidered therapeutically beneficial. Thus, in some embodiments, anamount of the bispecific antibody that provides a physiological changeis considered an “effective amount” or a “therapeutically effectiveamount”.

In a further aspect, the invention provides a method for treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of bispecific antibodies comprising afirst antigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM-3 of the invention.In one embodiment a composition is administered to said individual,comprising a bispecific antibody of the invention in a pharmaceuticallyacceptable form. In certain embodiments the disease to be treated is aproliferative disorder. In a particular embodiment the disease iscancer. In certain embodiments the method further comprisesadministering to the individual a therapeutically effective amount of atleast one additional therapeutic agent, e.g. an anti-cancer agent if thedisease to be treated is cancer. In another aspect, the disease is achronic viral infection. An “individual” according to any of the aboveembodiments may be a mammal, preferably a human.

For the prevention or treatment of disease, the appropriate dosage of abispecific antibodies comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM-3 of the invention (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the route ofadministration, the body weight of the patient, the type of fusionprotein, the severity and course of the disease, whether the bispecificantibody is administered for preventive or therapeutic purposes,previous or concurrent therapeutic interventions, the patient's clinicalhistory and response to the fusion protein, and the discretion of theattending physician. The practitioner responsible for administrationwill, in any event, determine the concentration of active ingredient(s)in a composition and appropriate dose(s) for the individual subject.Various dosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

The bispecific antibody comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM-3 as defined herein is suitably administeredto the patient at one time or over a series of treatments. Depending onthe type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g.0.1 mg/kg-10 mg/kg) of the bispecific antibody can be an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment would generally be sustained until a desired suppressionof disease symptoms occurs. One exemplary dosage of the bispecificantibody would be in the range from about 0.005 mg/kg to about 10 mg/kg.In other examples, a dose may also comprise from about 1 μg/kg bodyweight, about 5 g/kg body weight, about 10 μg/kg body weight, about 50μg/kg body weight, about 100 μg/kg body weight, about 200 μg/kg bodyweight, about 350 μg/kg body weight, about 500 μg/kg body weight, about1 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg bodyweight, about 50 mg/kg body weight, about 100 mg/kg body weight, about200 mg/kg body weight, about 350 mg/kg body weight, about 500 mg/kg bodyweight, to about 1000 mg/kg body weight or more per administration, andany range derivable therein. In examples of a derivable range from thenumbers listed herein, a range of about 5 mg/kg body weight to about 100mg/kg body weight, about 5 μg/kg body weight to about 500 mg/kg bodyweight etc., can be administered, based on the numbers described above.Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient.Such doses may be administered intermittently, e.g. every week or everythree weeks (e.g. such that the patient receives from about two to abouttwenty, or e.g. about six doses of the fusion protein). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

The bispecific antibodies comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM-3 as defined herein will generally be used inan amount effective to achieve the intended purpose. For use to treat orprevent a disease condition, the bispecific antibodies of the invention,or pharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the bispecific antibody which are sufficient tomaintain therapeutic effect. Usual patient dosages for administration byinjection range from about 0.1 to 50 mg/kg/day, typically from about 0.5to 1 mg/kg/day. Therapeutically effective plasma levels may be achievedby administering multiple doses each day. Levels in plasma may bemeasured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the bispecific antibody may not be related toplasma concentration. One skilled in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the bispecific antibodies describedherein will generally provide therapeutic benefit without causingsubstantial toxicity. Toxicity and therapeutic efficacy of a fusionprotein can be determined by standard pharmaceutical procedures in cellculture or experimental animals. Cell culture assays and animal studiescan be used to determine the LD₅₀ (the dose lethal to 50% of apopulation) and the ED₅₀ (the dose therapeutically effective in 50% of apopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀.Bispecific antibodies that exhibit large therapeutic indices arepreferred. In one embodiment, the bispecific antibody according to thepresent invention exhibits a high therapeutic index. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosages suitable for use in humans. The dosage lies preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage may vary within this range dependingupon a variety of factors, e.g., the dosage form employed, the route ofadministration utilized, the condition of the subject, and the like. Theexact formulation, route of administration and dosage can be chosen bythe individual physician in view of the patient's condition (see, e.g.,Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch.1, p. 1, incorporated herein by reference in its entirety).

The attending physician for patients treated with bispecific antibodiesof the invention would know how and when to terminate, interrupt, oradjust administration due to toxicity, organ dysfunction, and the like.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated, with the route of administration, and the like. The severityof the condition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and perhaps dosefrequency will also vary according to the age, body weight, and responseof the individual patient.

Other Agents and Treatments

The bispecific antibodies comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM-3 as described herein before may beadministered in combination with one or more other agents in therapy.For instance, a fusion protein of the invention may be co-administeredwith at least one additional therapeutic agent. The term “therapeuticagent” encompasses any agent that can be administered for treating asymptom or disease in an individual in need of such treatment. Suchadditional therapeutic agent may comprise any active ingredientssuitable for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.In certain embodiments, an additional therapeutic agent is anotheranti-cancer agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of fusion protein used, the type ofdisorder or treatment, and other factors discussed above. The TNF familyligand trimer-containing antigen binding molecules are generally used inthe same dosages and with administration routes as described herein, orabout from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the bispecific antibody can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper that ispierceable by a hypodermic injection needle). At least one active agentin the composition is a bispecific antibody comprising a firstantigen-binding site that specifically binds to PD1 and a secondantigen-binding site that specifically binds to TIM-3 as defined hereinbefore.

The label or package insert indicates that the composition is used fortreating the condition of choice. Moreover, the article of manufacturemay comprise (a) a first container with a composition contained therein,wherein the composition comprises the bispecific antibody of theinvention; and (b) a second container with a composition containedtherein, wherein the composition comprises a further cytotoxic orotherwise therapeutic agent. The article of manufacture in thisembodiment of the invention may further comprise a package insertindicating that the compositions can be used to treat a particularcondition.

Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

TABLE C  (Sequences): SEQ ID NO: Name Sequence  1heavy chain HVR-H1, Tim3_0016 GFSLSTSGM  2 heavy chain HVR-H2, Tim3_0016LND  3 heavy chain HVR-H3, Tim3_0016 NGYLYALD  4light chain HVR-L1, Tim3_0016 SSSVNY  5 light chain HVR-L2, Tim3_0016DAF  6 light chain HVR-L3, Tim3_0016 WSSYPWT  7heavy chain variable domain VH, QVTLKESGPG ILQPSQTLRL TCSFSGFSLSTim3_0016 TSGMSVGWIR QPSGKGLEWL AHIWLNDDVFFNPALKSRLT ISKDTSNNQV FLQIASVVTA DTATYYCVRA NGYLYALDYW GQGTSVTVSS  8light chain variable domain VL, QIVLTQSPAI MSASPGQKVT ITCSASSSVNTim3_0016 YTQWYQQKLG SSPKLWIYDA FKLAPGVPARFSGSGTGTSY SLTISSMEAE DAASYFCHQW SSYPWTFGGG TKLEIK  9heavy chain variable domain VH, QVTLKESGPG ILQPSQTLSL TCSFSGFSLSTim3_0016 variant (0018) TSGMSVGWIR QPSGKGLEWL AHIWLNDDVFFNPALKRRLT ISKDTSNNQV FLQIASVVTA DTATYYCVRA NGYLYALDYW GQGISVTVSS 10light chain variable domain VL, QIVLTQSPAI MSASPGQKVT ITCSASSSVNTim3_0016 variant (0018) YTQWYQQKLG SSPKLWIYDA FKLAPGVPARFSGSGTGTSY SLTISSMEAE DAASYFCHQW SSYPWTFGGG TKLEIK 11light chain HVR-L1, Tim3_0016_HVR-L1 SSSVQY variant 1_NQ(removal of glycosylation site by N to Q mutation) 12light chain HVR-L1, Tim3_0016_HVR-L1 SSSVSY variant 2_NS(removal of glycosylation site by N to S mutation) 13VH humanized version of Tim3_0016 QITLKESGPT LVKPTQTLTL TCTFSGFSLSvariant (0018) (=Tim3-0433) TSGMSVGWIR QPPGKGLEWL AHIWLNDDVFFNPALKSRLT ITKDTSKNQV VLTMTNMDPV DTATYYCVRA NGYLYALDYW GQGTLVTVSS 14VL humanized version of Tim3_0016 ETTLTQSPAF MSATPGDKVN IACSASSSVSvariant (0018) (=Tim3-0433) YTQWYQQKPG EAPKLWIYDA FKLAPGIPPRFSGSGYGTDF TLTINNIESE DAAYYFCHQW SSYPWTFGQG TKLEIK 15VH humanized version of Tim3_0016 QITLKESGPT LVKPTQTLTL TCTFSGFSLSvariant (0018) (=Tim3-0434) TSGMSVGWIR QPPGKGLEWL AHIWLNDDVFFNPALKSRLT ITKDTSKNQV VLTMTNMDPV DTATYYCVRA NGYLYALDYW GQGTLVTVSS 16VL humanized version of Tim3_0016 DIQLTQSPSF LSASVGDRVT ITCSASSSVSvariant (0018) (=Tim3-0434) YTQWYQQKPG KAPKLWIYDA FKLAPGVPSR 60FSGSGSGTEF TLTISSLQPE DFATYFCHQW SSYPWTFGQG TKLEIK 17heavy chain HVR-H1, Tim3_0028 GFNIKTT 18 heavy chain HVR-H2, Tim3_0028ADD 19 heavy chain HVR-H3, Tim3_0028 FGYVAWFA 20light chain HVR-L1, Tim3_0028 SQSVDNY 21 light chain HVR-L2, Tim3_0028YAS 22 light chain HVR-L3, Tim3_0028 HYSSPY 23heavy chain variable domain VH, EVQLQQSVAE LVRPGASVKL SCTASGFNIKTim3_0028 TTYMHWVKQR PEQGLEWIGR IDPADDNTKYAPKFQGKATI TADTSSNTAY LQLSSLTSED AAIYYCVRDF GYVAWFAYWG QGTLVTFSA 24light chain variable domain VL, NIVMTPTPKF LPVSSGDRVT MTCRASQSVDTim3_0028 NYVAWYQQKP GQSPKLLIYY ASNRYIGVPDRFTGSGSGTD FTFTISSVQV EDLAVYFCQQ HYSSPYTFGS GTKLEIK 25VH humanized version of Tim3-0028  EVQLVESGGG LVQPGGSLRL SCAASGFNIK(=Tim3-0438) TTYMHWVRQA PGKGLEWVGR IDPADDNTKYAPKFQGKATI SADTSKNTAY LQMNSLRAED TAVYYCVRDF GYVAWFAYWG QGTLVTVSS 26VL humanized version of Tim3-0028  DIVMTQSPLS LPVTPGEPAS ISCRASQSVD(=Tim3-0438) NYVAWYLQKP GQSPQLLIYY ASNRYIGVPDRFSGSGSGTD FTLKISRVEA EDVGVYYCQQ HYSSPYTFGQ GTKVEIK 27VH humanized version of Tim3-0028 EVQLVESGGG LVQPGGSLRL SCAASGFNIK(=Tim3-0443) TTYMHWVRQA PGKGLEWVGR IDPADDNTKYAPKFQGKATI SADTSKNTAY LQMNSLRAED TAVYYCVRDF GYVAWFAYWG QGTLVTFSS 28VL humanized version of Tim3-0028  DIVMTQSPLS LPVTPGEPAS ISCRASQSVD(=Tim3-0443) NYVAWYLQKP GQSPQLLIYY ASNRYIGVPDRFSGSGSGTD FTLKISRVEA EDVGVYYCQQ HYSSPYTFGQ GTKVEIK 29heavy chain HVR-H1, Tim3_0038 GFNIKDY 30 heavy chain HVR-H2, Tim3_0038EDG 31 heavy chain HVR-H3, Tim3_0038 HGYVGWFA 32light chain HVR-L1, Tim3_0038 ASENVDTY 33 light chain HVR-L2, Tim3_0038GAS 34 light chain HVR-L3, Tim3_0038 SYSYPW 35heavy chain variable domain VH, EVQLQQSGAE PLKPGASVKL TCTTSGFNIKTim3_0038 DYYIHWVKQR SDQGLEWIGR IDPEDGELIYAPKFQDKATI TVDTSSNIAY LQLNSLTSED TAVYYCSRDH GYVGWFAYWG QGTLVTVSA 36light chain variable domain VL, NVVMTQSPKS MIMSVGQRVT LNCKASENVDTim3_0038 TYVSWYQQKP EQSPKLLIYG ASNRYTGVPDRFTGSRSATD FTLTISSVQA EDLAVYYCGQ SYSYPWTFGG GTKLEFR 37heavy chain HVR-H1, PD1-0103 GFSFSSY 38 heavy chain HVR-H2, PD1-0103 GGR39 heavy chain HVR-H3, PD1-0103 TGRVYFALD 40light chain HVR-L1, PD1-0103 SESVDTSDNSF 41 light chain HVR-L2, PD1-0103RSS 42 light chain HVR-L3, PD1-0103 NYDVPW 43heavy chain variable domain VH, PD1- EVILVESGGGLVKPGGSLKLSCAASGFSFSSY0103 TMSWVRQTPEKRLDWVATISGGGRDIYYPDSV KGRFTISRDNAKNTLYLEMSSLMSEDTALYYCVLLTGRVYFALDSWGQGTSVTVSS 44 light chain variable domain VL, PD1-0103KIVLTQSPASLPVSLGQRATISCRASESVDTS DNSFIHWYQQRPGQSPKLLIYRSSTLESGVPARFSGSGSRTDFTLTIDPVEADDVATYYCQQNY DVPWTFGGGTKLEIK 45humanized variant -heavy chain variable EVQLLESGGGLVQPGGSLRLSCAASGFSFSSYdomain VH of PD1-0103_01 TMSWVRQAPGKGLEWVATISGGGRDIYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC VLLTGRVYFALDSWGQGTLVTVSS 46humanized variant -light chain variable DIVMTQSPDSLAVSLGERATINCKASESVDTSdomain VL of PD1-0103_01 DNSFIHWYQQKPGQSPKLLIYRSSTLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNY DVPWTFGQGTKVEIK 47humanized variant -light chain variable DVVMTQSPLSLPVTLGQPASISCRASESVDTSdomain VL of PD1-0103_02 DNSFIHWYQQRPGQSPRLLIYRSSTLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQNY DVPWTFGQGTKVEIK 48humanized variant -light chain variable EIVLTQSPATLSLSPGERATLSCRASESVDTSdomain VL of PD1-0103_03 DNSFIHWYQQKPGQSPRLLIYRSSTLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQNY DVPWTFGQGTKVEIK 49humanized variant -light chain variable EIVLTQSPATLSLSPGERATLSCRASESVDTSdomain VL of PD1-0103_04 DNSFIHWYQQKPGQSPRLLIYRSSTLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQNY DVPWTFGQGTKVEIK 50heavy chain 1 of 1 + 1 PD1TIM3_0389 KIVLTQSPAS LPVSLGQRAT ISCRASESVD(based on chimeric PD1-0103/Tim3- TSDNSFIHWY QQRPGQSPKL LIYRSSTLES 0028)GVPARFSGSG SRTDFTLTID PVEADDVATY YCQQNYDVPW TFGGGTKLEI KSSASTKGPSVFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSSVVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEA AGGPSVFLFPPKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVSVLTVLHQDWL NGKEYKCKVS NKALGAPIEK TISKAKGQPR EPQVYTLPPC RDELTKNQVSLWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFSCSVMHEALHN HYTQKSLSLS PGK 51 heavy chain 2 of 1 + 1 PD1TIM3_0389EVQLQQSVAE LVRPGASVKL SCTASGFNIK TTYMHWVKQR PEQGLEWIGR IDPADDNTKYAPKFQGKATI TADTSSNTAY LQLSSLTSED AAIYYCVRDF GYVAWFAYWG QGTLVTFSAASTKGPSVFPL APSSKSTSGG TAALGCLVED YFPEPVTVSW NSGALTSGVH TFPAVLQSSGLYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDEKVEPK SCDKTHTCPP CPAPEAAGGPSVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKAL GAPIEKTISK AKGQPREPQV CTLPPSRDELTKNQVSLSCA VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLVS KLTVDKSRWQQGNVFSCSVM HEALHNRFTQ KSLSLSPGK 52 light chain 1 of 1 + 1 PD1TIM3_0389EVILVESGGG LVKPGGSLKL SCAASGFSFS SYTMSWVRQT PEKRLDWVAT ISGGGRDIYYPDSVKGRFTI SRDNAKNTLY LEMSSLMSED TALYYCVLLT GRVYFALDSW GQGTSVTVSSASVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQDSKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 53light chain 2 of 1 + 1 PD1TIM3_0389 NIVMTPTPKF LPVSSGDRVT MTCRASQSVDNYVAWYQQKP GQSPKLLIYY ASNRYIGVPD RFTGSGSGTD FTFTISSVQV EDLAVYFCQQHYSSPYTFGS GTKLEIKRTV AAPSVFIFPP SDRKLKSGTA SVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC54 heavy chain 1 of 1 + 1 PD1TIM3_0168 KIVLTQSPAS LPVSLGQRAT ISCRASESVD(based on chimeric PD1-0103/Tim3- TSDNSFIHWY QQRPGQSPKL LIYRSSTLES 0018)GVPARFSGSG SRTDFTLTID PVEADDVATY YCQQNYDVPW TFGGGTKLEI KSSASTKGPSVFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSSVVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEA AGGPSVFLFPPKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVSVLTVLHQDWL NGKEYKCKVS NKALGAPIEK TISKAKGQPR EPQVYTLPPC RDELTKNQVSLWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFSCSVMHEALHN HYTQKSLSLS PGK 55 heavy chain 2 of 1 + 1 PD1TIM3_0168QVTLKESGPG ILQPSQTLSL TCSFSGFSLS TSGMSVGWIR QPSGKGLEWL AHIWLNDDVFFNPALKRRLT ISKDTSNNQV FLQIASVVTA DTATYYCVRA NGYLYALDYW GQGISVTVSSASTKGPSVFP LAPSSKSTSG GTAALGCLVE DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDEKVEP KSCDKTHTCP PCPAPEAAGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LGAPIEKTIS KAKGQPREPQ VCTLPPSRDELTKNQVSLSC AVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLV SKLTVDKSRWQQGNVFSCSV MHEALHNRFT QKSLSLSPGK 56 light chain 1 of 1 + 1 PD1TIM3_0168EVILVESGGG LVKPGGSLKL SCAASGFSFS SYTMSWVRQT PEKRLDWVAT ISGGGRDIYYPDSVKGRFTI SRDNAKNTLY LEMSSLMSED TALYYCVLLT GRVYFALDSW GQGTSVTVSSASVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQDSKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 57light chain 2 of 1 + 1 PD1TIM3_0168 QIVLTQSPAI MSASPGQKVT ITCSASSSVNYTQWYQQKLG SSPKLWIYDA FKLAPGVPAR FSGSGTGTSY SLTISSMEAE DAASYFCHQWSSYPWTFGGG TKLEIKRTVA APSVFIFPPS DRKLKSGTAS VVCLLNNFYP REAKVQWKVDNALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 58heavy chain 1 of 1 + 1 PD1TIM3_0166: KIVLTQSPAS LPVSLGQRAT ISCRASESVD(based on chimeric PD1-0103/Tim3-0038 TSDNSFIHWY QQRPGQSPKL LIYRSSTLESGVPARFSGSG SRTDFTLTID PVEADDVATY YCQQNYDVPW TFGGGTKLEI KSSASTKGPSVFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSSVVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEA AGGPSVFLFPPKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVSVLTVLHQDWL NGKEYKCKVS NKALGAPIEK TISKAKGQPR EPQVYTLPPC RDELTKNQVSLWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFSCSVMHEALHN HYTQKSLSLS PGK 59 heavy chain 2 of 1 + 1 PD1TIM3_0166EVQLQQSGAE PLKPGASVKL TCTTSGFNIK DYYIHWVKQR SDQGLEWIGR IDPEDGELIYAPKFQDKATI TVDTSSNIAY LQLNSLTSED TAVYYCSRDH GYVGWFAYWG QGTLVTVSAASTKGPSVFPL APSSKSTSGG TAALGCLVED YFPEPVTVSW NSGALTSGVH TFPAVLQSSGLYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDEKVEPK SCDKTHTCPP CPAPEAAGGPSVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKAL GAPIEKTISK AKGQPREPQV CTLPPSRDELTKNQVSLSCA VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLVS KLTVDKSRWQQGNVFSCSVM HEALHNRFTQ KSLSLSPGK 60 light chain 1 of 1 + 1 PD1TIM3_0166EVILVESGGG LVKPGGSLKL SCAASGFSFS SYTMSWVRQT PEKRLDWVAT ISGGGRDIYYPDSVKGRFTI SRDNAKNTLY LEMSSLMSED TALYYCVLLT GRVYFALDSW GQGTSVTVSSASVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQDSKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 61light chain 2 of 1 + 1 PD1TIM3_0166 NVVMTQSPKS MIMSVGQRVT LNCKASENVDTYVSWYQQKP EQSPKLLIYG ASNRYTGVPD RFTGSRSATD FTLTISSVQA EDLAVYYCGQSYSYPWTFGG GTKLEFRRTV AAPSVFIFPP SDRKLKSGTA SVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC62 heavy chain 1 of 1 + 1 PD1TIM3_0476: DIVMTQSPDS LAVSLGERAT INCKASESVD(based on humanized PD1-0103_0312)/ TSDNSFIHWY QQKPGQSPKL LIYRSSTLESTim3-0438) GVPDRFSGSG SGTDFTLTIS SLQAEDVAVYYCQQNYDVPW TFGQGTKVEI KSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPVTVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKKVEPKSCDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVKFNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALGAPIEKTISKAKGQPR EPQVYTLPPC RDELTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 63heavy chain 2 of 1 + 1 PD1TIM3_0476 EVQLVESGGG LVQPGGSLRL SCAASGFNIKTTYMHWVRQA PGKGLEWVGR IDPADDNTKY APKFQGKATI SADTSKNTAY LQMNSLRAEDTAVYYCVRDF GYVAWFAYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVEDYFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSNTKVDEKVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSHEDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKALGAPIEKTISK AKGQPREPQV CTLPPSRDEL TKNQVSLSCA VKGFYPSDIA VEWESNGQPENNYKTTPPVL DSDGSFFLVS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGK 64light chain 1 of 1 + 1 PD1TIM3_0476 EVQLLESGGG LVQPGGSLRL SCAASGFSFSSYTMSWVRQA PGKGLEWVAT ISGGGRDIYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCVLLT GRVYFALDSW GQGTLVTVSS ASVAAPSVFI FPPSDEQLKS GTASVVCLLNNFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGEC 65 light chain 2 of 1 + 1 PD1TIM3_0476DIVMTQSPLS LPVTPGEPAS ISCRASQSVD NYVAWYLQKP GQSPQLLIYY ASNRYIGVPDRFSGSGSGTD FTLKISRVEA EDVGVYYCQQ HYSSPYTFGQ GTKVEIKRTV AAPSVFIFPPSDRKLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLTLSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 66heavy chain 1 of 1 + 1 PD1TIM3_0477: DIVMTQSPDS LAVSLGERAT INCKASESVD(based on humanized PD1-0103_0312)/ TSDNSFIHWY QQKPGQSPKL LIYRSSTLESTim3-0434) GVPDRFSGSG SGTDFTLTIS SLQAEDVAVYYCQQNYDVPW TFGQGTKVEI KSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPVTVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKKVEPKSCDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVKFNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALGAPIEKTISKAKGQPR EPQVYTLPPC RDELTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK 67heavy chain 2 of 1 + 1 PD1TIM3_0477 QITLKESGPT LVKPTQTLTL TCTFSGFSLSTSGMSVGWIR QPPGKGLEWL AHIWLNDDVF FNPALKSRLT ITKDTSKNQV VLTMTNMDPVDTATYYCVRA NGYLYALDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVEDYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPSNTKVDEKVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKALGAPIEKTIS KAKGQPREPQ VCTLPPSRDE LTKNQVSLSC AVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLV SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 68light chain 1 of 1 + 1 PD1TIM3_0477 EVQLLESGGG LVQPGGSLRL SCAASGFSFSSYTMSWVRQA PGKGLEWVAT ISGGGRDIYY PDSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCVLLT GRVYFALDSW GQGTLVTVSS ASVAAPSVFI FPPSDEQLKS GTASVVCLLNNFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGEC 69 light chain 2 of 1 + 1 PD1TIM3_0477DIQLTQSPSF LSASVGDRVT ITCSASSSVS YTQWYQQKPG KAPKLWIYDA FKLAPGVPSRFSGSGSGTEF TLTISSLQPE DFATYFCHQW SSYPWTFGQG TKLEIKRTVA APSVFIFPPSDRKLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTLSKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 70heavy chain of 2 + 2 PD1TIM3_0358: EVQLQQSVAE LVRPGASVKL SCTASGFNIKchimeric PD1-0103/Tim3-0028 TTYMHWVKQR PEQGLEWIGR IDPADDNTKYAPKFQGKATI TADTSSNTAY LQLSSLTSED AAIYYCVRDF GYVAWFAYWG QGTLVTFSAASTKGPSVFPL APSSKSTSGG TAALGCLVED YFPEPVTVSW NSGALTSGVH TFPAVLQSSGLYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDEKVEPK SCDKTHTCPP CPAPEAAGGPSVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNSTYRVVSVLTV LHQDWLNGKE YKCKVSNKAL GAPIEKTISK AKGQPREPQV YTLPPSRDELTKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQQGNVFSCSVM HEALHNHYTQ KSLSLSPGGG GGSGGGGSGG GGSGGGGSKI VLTQSPASLPVSLGQRATIS CRASESVDTS DNSFIHWYQQ RPGQSPKLLI YRSSTLESGV PARFSGSGSRTDFTLTIDPV EADDVATYYC QQNYDVPWTF GGGTKLEIKS SASTKGPSVF PLAPSSKSTSGGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQTYICNVNHKP SNTKVDKKVE PKSCD 71 light chain 1 of 2 + 2 PD1TIM3_0358NIVMTPTPKF LPVSSGDRVT MTCRASQSVD NYVAWYQQKP GQSPKLLIYY ASNRYIGVPDRFTGSGSGTD FTFTISSVQV EDLAVYFCQQ HYSSPYTFGS GTKLEIKRTV AAPSVFIFPPSDRKLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLTLSKADYEKHK VYACEVTHQG 72 light chain 2 of 2 + 2 PD1TIM3_0358EVILVESGGG LVKPGGSLKL SCAASGFSFS SYTMSWVRQT PEKRLDWVAT ISGGGRDIYYPDSVKGRFTI SRDNAKNTLY LEMSSLMSED TALYYCVLLT GRVYFALDSW GQGTSVTVSSASVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQDSKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 73heavy chain of 2 + 2 PD1TIM3_0359: QVTLKESGPG ILQPSQTLSL TCSFSGFSLSchimeric PD1-0103/Tim3-0018 TSGMSVGWIR QPSGKGLEWL AHIWLNDDVFFNPALKRRLT ISKDTSNNQV FLQIASVVTA DTATYYCVRA NGYLYALDYW GQGISVTVSSASTKGPSVFP LAPSSKSTSG GTAALGCLVE DYFPEPVTVS WNSGALTSGV HTFPAVLQSSGLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDEKVEP KSCDKTHTCP PCPAPEAAGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LGAPIEKTIS KAKGQPREPQ VYTLPPSRDELTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGG GGGSGGGGSG GGGSGGGGSK IVLTQSPASLPVSLGQRATI SCRASESVDT SDNSFIHWYQ QRPGQSPKLL IYRSSTLESG VPARFSGSGSRTDFTLTIDP VEADDVATYY CQQNYDVPWT FGGGTKLEIK SSASTKGPSV FPLAPSSKSTSGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTQTYICNVNHK PSNTKVDKKV EPKSCD 74 light chain 1 of 2 + 2 PD1TIM3_0359QIVLTQSPAI MSASPGQKVT ITCSASSSVN YTQWYQQKLG SSPKLWIYDA FKLAPGVPARFSGSGTGTSY SLTISSMEAE DAASYFCHQW SSYPWTFGGG TKLEIKRTVA APSVFIFPPSDRKLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTLSKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 75light chain 2 of 2 + 2 PD1TIM3_0359 EVILVESGGG LVKPGGSLKL SCAASGFSFSSYTMSWVRQT PEKRLDWVAT ISGGGRDIYY PDSVKGRFTI SRDNAKNTLY LEMSSLMSEDTALYYCVLLT GRVYFALDSW GQGTSVTVSS ASVAAPSVFI FPPSDEQLKS GTASVVCLLNNFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGEC 76 heavy chain of 2 + 2 PD1TIM3_0321:EVQLQQSGAE PLKPGASVKL TCTTSGFNIK chimeric PD1-0103/Tim3-0038DYYIHWVKQR SDQGLEWIGR IDPEDGELIY APKFQDKATI TVDTSSNIAY LQLNSLTSEDTAVYYCSRDH GYVGWFAYWG QGTLVTVSAA STKGPSVFPL APSSKSTSGG TAALGCLVEDYFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV PSSSLGTQTY ICNVNHKPSNTKVDEKVEPK SCDKTHTCPP CPAPEAAGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSHEDPEVKFNWY VDGVEVHNAK TKPREEQYNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKALGAPIEKTISK AKGQPREPQV YTLPPSRDEL TKNQVSLTCL VKGFYPSDIA VEWESNGQPENNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ QGNVFSCSVM HEALHNHYTQ KSLSLSPGGGGGSGGGGSGG GGSGGGGSKI VLTQSPASLP VSLGQRATIS CRASESVDTS DNSFIHWYQQRPGQSPKLLI YRSSTLESGV PARFSGSGSR TDFTLTIDPV EADDVATYYC QQNYDVPWTFGGGTKLEIKS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSGVHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCD 77light chain 1 of 2 + 2 PD1TIM3_0321 NVVMTQSPKS MIMSVGQRVT LNCKASENVDTYVSWYQQKP EQSPKLLIYG ASNRYTGVPD RFTGSRSATD FTLTISSVQA EDLAVYYCGQSYSYPWTFGG GTKLEFRRTV AAPSVFIFPP SDRKLKSGTA SVVCLLNNFY PREAKVQWKVDNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC78 light chain 2 of 2 + 2 PD1TIM3_0321 EVILVESGGG LVKPGGSLKL SCAASGFSFSSYTMSWVRQT PEKRLDWVAT ISGGGRDIYY PDSVKGRFTI SRDNAKNTLY LEMSSLMSEDTALYYCVLLT GRVYFALDSW GQGTSVTVSS ASVAAPSVFI FPPSDEQLKS GTASVVCLLNNFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVTHQGLSSPVTK SFNRGEC 79 human kappa light chain constant regionRTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQDSKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC 80human lambda light chain constant regionQPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA GVETTTPSKQSNNKYAASSY LSLTPEQWKS HRSYSCQVTH EGSTVEKTVA PTECS 81human heavy chain constant region derivedASTKGPSVFP LAPSSKSTSG GTAALGCLVK from IgG1DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 82human heavy chain constant region derivedASTKGPSVFP LAPSSKSTSG GTAALGCLVK from IgG1 with mutations L234A andDYFPEPVTVS WNSGALTSGV HTFPAVLQSS L235A GLYSLSSVVT VPSSSLGTQT YICNVNHKPSNTKVDKKVEP KSCDKTHTCP PCPAPEAAGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVSHEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKALPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQPENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 83human heavy chain constant region derivedASTKGPSVFP LAPSSKSTSG GTAALGCLVK from IgG1 with mutations L234A, L235ADYFPEPVTVS WNSGALTSGV HTFPAVLQSS and P329GGLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKA LGAPIEKTIS KAKGQPREPQ VYTLPPSRDELTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRWQQGNVFSCSV MHEALHNHYT QKSLSLSPGK 84human heavy chain constant region derivedASTKGPSVFP LAPCSRSTSE STAALGCLVK from IgG4DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT YTCNVDHKPSNTKVDKRVES KYGPPCPSCP APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQEDPEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPSSIEKTISKAK GQPREPQVYT LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENNYKTTPPVLDS DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK 85exemplary human TIM3 sequences SEVEYRAEVG QNAYLPCFYT PAAPGNLVPVCWGKGACPVF ECGNVVLRTD ERDVNYWTSR YWLNGDFRKG DVSLTIENVT LADSGIYCCRIQIPGIMNDE KFNLKLVIKP AKVTPAPTRQ RDFTAAFPRM LTTRGHGPAE TQTLGSLPDINLTQISTLAN ELRDSRLAND LRDSGATIRI GIYIGAGICA GLALALIFGA LIFKWYSHSKEKIQNLSLIS LANLPPSGLA NAVAEGIRSE ENIYTIEENV YEVEEPNEYY CYVSSRQQPSQPLGCRFAMP 86 human TIM3 Extracellular Domain (ECD)SEVEYRAEVG QNAYLPCFYT PAAPGNLVPV CWGKGACPVF ECGNVVLRTD ERDVNYWTSRYWLNGDFRKG DVSLTIENVT LADSGIYCCR IQIPGIMNDE KFNLKLVIKP AKVTPAPTRQRDFTAAFPRM LTTRGHGPAE TQTLGSLPDI NLTQISTLAN ELRDSRLAND LRDSGATIRI G 87exemplary human PD1 sequence PGWFLDSPDR PWNPPTFSPA LLVVTEGDNATFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSVVRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLVVGVVGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQWREKTPEPPVP CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL 88human PD1 Extracellular Domain (ECD) PGWFLDSPDR PWNPPTFSPA LLVVTEGDNATFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSVVRARRNDSGT YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV 89human PD1 Extracellular Domain (ECD) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWincluding the signal peptide NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFR VTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSP RPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYG ELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL 90 GGGGS spacer GGGGS 91 SNAP-tagDKDCEMKRTTLDSPLGKLELSGCEQGLHEIKL LGKGTSAADAVEVPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQ VLWKLLKVVKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGG LAVKEWLLAHEGHRLGKPGLGPAGGSPGLEVN 92Flag-tag DYKDDDDK 93 human TIM3 Extracellular Domain (ECD)MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQ including signal peptideNAYLPCFYTPAAPGNLVPVCWGKGACPVFECG NVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLV IKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDL RDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAE GIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAM 94 Clip-tag DKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPAAVLGGPEPLIQATAW LNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISESHLAALVGNPAATAA VNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLAVKEWLLAHEGHRLGKPGLG

In the following specific embodiments of the invention are listed:

-   1. A bispecific antibody comprising a first antigen-binding site    that specifically binds to PD1 and a second antigen-binding site    that specifically binds to TIM3, wherein    -   said first antigen-binding site specifically binding to PD1        comprises        -   a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:37,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:            38, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:39; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:40;        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:41, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:42; and    -   said second antigen-binding site specifically binding to TIM3        comprises    -   (a) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:1,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:2, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:3; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4            or SEQ ID NO:11 or SEQ ID NO:12,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:5, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:6; or    -   (b) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:17,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:18, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:19; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:20,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:21, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:22; or    -   (c) a VH domain comprising        -   (i) HVR-H1 comprising the amino acid sequence of SEQ ID            NO:29,        -   (ii) HVR-H2 comprising the amino acid sequence of SEQ ID            NO:30, and        -   (iii) HVR-H3 comprising an amino acid sequence of SEQ ID            NO:31; and        -   a VL domain comprising        -   (i) HVR-L1 comprising the amino acid sequence of SEQ ID            NO:32,        -   (ii) HVR-L2 comprising the amino acid sequence of SEQ ID            NO:33, and        -   (iii) HVR-L3 comprising the amino acid sequence of SEQ ID            NO:34.-   2. A bispecific antibody comprising a first antigen-binding site    that specifically binds to PD1 and a second antigen-binding site    that specifically binds to TIM3, wherein the bispecific antibody    binds to TIM3 with an at least 50 fold lower binding affinity when    compared to the binding to PD1, more particularly with an at least    100 fold lower binding affinity when compared to the binding to PD1.-   3. The bispecific antibody according as defined herein before,    wherein    -   said first antigen-binding site specifically binding to PD1        comprises        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 43 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 44, or        -   (b) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 46, or        -   (c) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 47, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 48, or        -   (e) a VH domain comprising the amino acid sequence of SEQ ID            NO: 45 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 49,    -   and said second antigen-binding site specifically binding to        TIM3 comprises        -   (a) a VH domain comprising the amino acid sequence of SEQ ID            NO: 7 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 8, or        -   (b) a VH domain comprising the amino acid sequence of SEQ ID            NO: 9 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 10, or        -   (c) a VH domain comprising the amino acid sequence of SEQ ID            NO: 13 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 14, or        -   (d) a VH domain comprising the amino acid sequence of SEQ ID            NO: 15 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 16, or        -   (e) a VH domain comprising the amino acid sequence of SEQ ID            NO: 23 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 24, or        -   (f) a VH domain comprising the amino acid sequence of SEQ ID            NO: 25 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 26, or        -   (g) a VH domain comprising the amino acid sequence of SEQ ID            NO: 27 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 28, or        -   (h) a VH domain comprising the amino acid sequence of SEQ ID            NO: 35 and a VL domain comprising the amino acid sequence of            SEQ ID NO: 36.-   4. The bispecific antibody as defined hereinbefore, wherein    -   said first antigen-binding site specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 45 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 46,    -   and said second antigen-binding site specifically binding to        TIM3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 15 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 16 or a VH domain comprising the amino acid        sequence of SEQ ID NO: 25 and a VL domain comprising the amino        acid sequence of SEQ ID NO: 26.-   5. The bispecific antibody as defined herein before, wherein    -   said first antigen-binding site specifically binding to PD1        comprises a VH domain comprising the amino acid sequence of SEQ        ID NO: 45 and a VL domain comprising the amino acid sequence of        SEQ ID NO: 46,    -   and said second antigen-binding site specifically binding to        TIM3 comprises a VH domain comprising the amino acid sequence of        SEQ ID NO: 25 and a VL domain comprising the amino acid sequence        of SEQ ID NO: 26.-   6. The bispecific antibody as defined herein before, wherein the    bispecific antibody is a human, humanized or chimeric antibody.-   7. The bispecific antibody as defined herein before, wherein the    bispecific antibody comprises an Fc domain, a first Fab fragment    comprising the antigen-binding site that specifically binds to PD1    and a second Fab fragment comprising the antigen-binding site that    specifically binds to TIM3.-   8. The bispecific antibody as defined herein before, wherein the Fc    domain is an IgG, particularly an IgG1 Fc domain or an IgG4 Fc    domain.-   9. The bispecific antibody as defined herein before, wherein the Fc    domain comprises one or more amino acid substitution that reduces    binding to an Fc receptor, in particular towards Fcγ receptor.-   10. The bispecific antibody as defined herein before, wherein the Fc    domain is of human IgG1 subclass with the amino acid mutations    L234A, L235A and P329G (numbering according to Kabat EU index).-   11. The bispecific antibody as defined herein before, wherein the Fc    domain comprises a modification promoting the association of the    first and second subunit of the Fc domain.-   12. The bispecific antibody as defined herein before, wherein the    first subunit of the Fc domain comprises knobs and the second    subunit of the Fc domain comprises holes according to the knobs into    holes method.-   13. The bispecific antibody as defined herein before, wherein the    first subunit of the Fc domain comprises the amino acid    substitutions S354C and T366W (EU numbering) and the second subunit    of the Fc domain comprises the amino acid substitutions Y349C, T366S    and Y407V (numbering according to Kabat EU index).-   14. The bispecific antibody as defined herein before, wherein in one    of the Fab fragments the variable domains VL and VH are replaced by    each other so that the VH domain is part of the light chain and the    VL domain is part of the heavy chain.-   15. The bispecific antibody as defined herein before, wherein in the    first Fab fragment comprising the antigen-binding site that    specifically binds to PD1 the variable domains VL and VH are    replaced by each other.-   16. The bispecific antibody as defined herein before, wherein in one    of the Fab fragments in the constant domain CL the amino acid at    position 124 is substituted independently by lysine (K),    arginine (R) or histidine (H) (numbering according to Kabat EU    Index), and in the constant domain CH1 the amino acids at positions    147 and 213 are substituted independently by glutamic acid (E) or    aspartic acid (D) (numbering according to Kabat EU index).-   17. The bispecific antibody as defined herein before, wherein in the    second Fab fragment comprising the antigen-binding site that    specifically binds to TIM3 the constant domain CL the amino acid at    position 124 is substituted independently by lysine (K),    arginine (R) or histidine (H) (numbering according to Kabat EU    Index), and in the constant domain CH1 the amino acids at positions    147 and 213 are substituted independently by glutamic acid (E) or    aspartic acid (D) (numbering according to Kabat EU index).-   18. The bispecific antibody as defined herein before, comprising-   (a) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 50, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 52, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 51, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:53, or-   (b) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 54, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 56, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 55, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:57, or-   (c) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 58, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 60, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 59, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:61, or-   (d) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 62, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 64, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 63, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:65, or-   (e) a first heavy chain comprising an amino acid sequence with at    least 95% sequence identity to the sequence of SEQ ID NO: 66, a    first light chain comprising an amino acid sequence with at least    95% sequence identity to the sequence of SEQ ID NO: 68, a second    heavy chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO: 67, and a second    light chain comprising an amino acid sequence with at least 95%    sequence identity to the sequence of SEQ ID NO:69.-   19. The bispecific antibody as defined herein before, comprising-   (a) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 50, a first light chain comprising the amino acid sequence of    SEQ ID NO: 52, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 51, and a second light chain comprising the    amino acid sequence of SEQ ID NO:53, or-   (b) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 54, a first light chain comprising the amino acid sequence of    SEQ ID NO: 56, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 55, and a second light chain comprising the    amino acid sequence of SEQ ID NO:57, or-   (c) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 58, a first light chain comprising the amino acid sequence of    SEQ ID NO: 60, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 59, and a second light chain comprising the    amino acid sequence of SEQ ID NO:61, or-   (d) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 62, a first light chain comprising the amino acid sequence of    SEQ ID NO: 64, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 63, and a second light chain comprising the    amino acid sequence of SEQ ID NO:65, or-   (e) a first heavy chain comprising the amino acid sequence of SEQ ID    NO: 66, a first light chain comprising the amino acid sequence of    SEQ ID NO: 68, a second heavy chain comprising the amino acid    sequence of SEQ ID NO: 67, and a second light chain comprising the    amino acid sequence of SEQ ID NO:69.-   20. A polynucleotide encoding the bispecific antibody as defined    herein before.-   21. A vector, particularly an expression vector, comprising the    polynucleotide as defined herein before.-   22. A prokaryotic or eukaryotic host cell comprising the    polynucleotide as defined herein before or the vector as defined    herein before.-   23. A method of producing the bispecific antibody as defined herein    before, comprising the steps of a) transforming a host cell with    vectors comprising polynucleotides encoding said bispecific    antibody, b) culturing the host cell according under conditions    suitable for the expression of the bispecific antibody and c)    recovering the bispecific antibody from the culture.-   24. A pharmaceutical composition comprising the bispecific antibody    as defined herein before and at least one pharmaceutically    acceptable excipient.-   25. The bispecific antibody as defined herein before or the    pharmaceutical composition as defined herein before for use as a    medicament.-   26. The bispecific antibody as defined herein before or the    pharmaceutical composition as defined herein before for use    -   i) in the modulation of immune responses, such as restoring T        cell activity,    -   ii) in stimulating an immune response or function,    -   iii) in the treatment of infections,    -   iv) in the treatment of cancer,    -   v) in delaying progression of cancer,    -   vi) in prolonging the survival of a patient suffering from        cancer.-   27. The bispecific antibody as defined herein before or the    pharmaceutical composition as defined herein before for use in the    prevention or treatment of cancer.-   28. The bispecific antibody as defined herein before or the    pharmaceutical composition as defined herein before for use in the    treatment of a chronic viral infection.-   29. The bispecific antibody as defined herein before or the    pharmaceutical composition as defined herein before for use in the    prevention or treatment of cancer, wherein the bispecific antibody    is administered in combination with a chemotherapeutic agent,    radiation and/or other agents for use in cancer immunotherapy.-   30. A method of inhibiting the growth of tumor cells in an    individual comprising administering to the individual an effective    amount of the bispecific antibody as defined herein before to    inhibit the growth of the tumor cells.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Materials & General Methods

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A., etal., Sequences of Proteins of Immunological Interest, 5th ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991).Amino acids of antibody chains are numbered and referred to according tothe numbering systems according to Kabat (Kabat, E. A., et al.,Sequences of Proteins of Immunological Interest, 5th ed., Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) as definedabove.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular Cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions.

Gene Synthesis

Desired gene segments were prepared from oligonucleotides made bychemical synthesis. The 600-1800 bp long gene segments, which wereflanked by singular restriction endonuclease cleavage sites, wereassembled by annealing and ligating oligonucleotides including PCRamplification and subsequently cloned via the indicated restrictionsites e.g. KpnI/SacI or AscI/PacI into a pPCRScript (Stratagene) basedpGA4 cloning vector. The DNA sequences of the subcloned gene fragmentswere confirmed by DNA sequencing. Gene synthesis fragments were orderedaccording to given specifications at Geneart (Regensburg, Germany).

DNA Sequence Determination

DNA sequences were determined by double strand sequencing performed atMediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH(Vaterstetten, Germany).

DNA and Protein Sequence Analysis and Sequence Data Management

The GCG's (Genetics Computer Group, Madison, Wis.) software packageversion 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was usedfor sequence creation, mapping, analysis, annotation and illustration.

Expression Vectors

For the expression of the described antibodies, variants of expressionplasmids for transient expression (e.g. in HEK293) cells based either ona cDNA organization with or without a CMV-Intron A promoter or on agenomic organization with a CMV promoter were applied.

Beside the antibody expression cassette the vectors contained:

-   -   an origin of replication which allows replication of this        plasmid in E. coli, and    -   a β-lactamase gene which confers ampicillin resistance in E.        coli.        The transcription unit of the antibody gene was composed of the        following elements:    -   unique restriction site(s) at the 5′ end    -   the immediate early enhancer and promoter from the human        cytomegalovirus,    -   followed by the Intron A sequence in the case of the cDNA        organization,    -   a 5′-untranslated region of a human antibody gene,    -   an immunoglobulin heavy chain signal sequence,    -   the human antibody chain (wildtype or with domain exchange)        either as cDNA or as genomic organization with the        immunoglobulin exon-intron organization    -   a 3′ untranslated region with a polyadenylation signal sequence,        and    -   unique restriction site(s) at the 3′ end.

The fusion genes comprising the antibody chains as described below weregenerated by PCR and/or gene synthesis and assembled by knownrecombinant methods and techniques by connection of the accordingnucleic acid segments e.g. using unique restriction sites in therespective vectors. The subcloned nucleic acid sequences were verifiedby DNA sequencing. For transient transfections larger quantities of theplasmids were prepared by plasmid preparation from transformed E. colicultures (Nucleobond AX, Macherey-Nagel).

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford,J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley &Sons, Inc.

Multispecific antibodies were expressed by transient co-transfection ofthe respective expression plasmids in adherently growing HEK293-EBNA orin HEK29-F cells growing in suspension as described below.

Transient Transfections in HEK293 System

All antibodies and bispecific antibodies were generated by transienttransfection of 293F cells using the Freestyle system (ThermoFisher).Here the 293F cells were cultivated in F17 Medium, transfected with293Free (Novagene) and feeded after 4 hours with VPA 4 mM and Feed 7 and0.6% Glucose after 16 h. Further the Expi293F™ Expression System Kit(ThermoFisher) was used. Here the Expi293F™ cells were cultivated inExpi293™ Expression Medium and transfected using ExpiFectamine™ 293Transfection Kit according manufacturer's instructions. Due to theimproved stability and purity and reduced aggregation tendency of theCrossMAb^(Vh-VL) bispecific antibodies with additionally introducedcharged pairs of amino acids in the CH1/CL interface (see positions inthe respective sequences for further detail) no adjustments of plasmidratio have been employed. Therefore the relative plasmid ratio of1:1:1:1 for 1+1 CrossMab or 1:1:1 for 2+2 CrossMab was used for theco-transfection of LC, HC, crossed LC and crossed HC plasmids. Cellsupernatants were harvested after 7 days and purified by standardmethods.

Protein Determination

The protein concentration of purified antibodies and derivatives wasdetermined by determining the optical density (OD) at 280 nm, using themolar extinction coefficient calculated on the basis of the amino acidsequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423.

Antibody Concentration Determination in Supernatants

The concentration of antibodies and derivatives in cell culturesupernatants was estimated by immunoprecipitation with Protein AAgarose-beads (Roche). 60 μL Protein A Agarose beads were washed threetimes in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40).Subsequently, 1-15 mL cell culture supernatant was applied to theProtein A Agarose beads pre-equilibrated in TBS-NP40. After incubationfor at 1 hour at room temperature the beads were washed on anUltrafree-MC-filter column (Amicon) once with 0.5 mL TBS-NP40, twicewith 0.5 mL 2× phosphate buffered saline (2×PBS, Roche) and briefly fourtimes with 0.5 mL 100 mM Na-citrate pH 5.0. Bound antibody was eluted byaddition of 35 μl NuPAGE® LDS Sample Buffer (Invitrogen). Half of thesample was combined with NuPAGE® Sample Reducing Agent or leftunreduced, respectively, and heated for 10 min at 70° C. Consequently,5-30 μl were applied to a 4-12% NuPAGE® Bis-Tris SDS-PAGE (Invitrogen)(with MOPS buffer for non-reduced SDS-PAGE and MES buffer with NuPAGE®Antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE)and stained with Coomassie Blue.

The concentration of antibodies and derivatives in cell culturesupernatants was quantitatively measured by affinity HPLCchromatography. Briefly, cell culture supernatants containing antibodiesand derivatives that bind to Protein A were applied to an AppliedBiosystems Poros A/20 column in 200 mM KH2PO4, 100 mM sodium citrate, pH7.4 and eluted from the matrix with 200 mM NaCl, 100 mM citric acid, pH2.5 on an Agilent HPLC 1100 system. The eluted protein was quantified byUV absorbance and integration of peak areas. A purified standard IgG1antibody served as a standard.

Alternatively, the concentration of antibodies and derivatives in cellculture supernatants was measured by Sandwich-IgG-ELISA. Briefly,StreptaWell High Bind Strepatavidin A-96 well microtiter plates (Roche)are coated with 100 μL/well biotinylated anti-human IgG capture moleculeF(ab′)2<h-Fcγ>BI (Dianova) at 0.1 μg/mL for 1 hour at room temperatureor alternatively overnight at 4° C. and subsequently washed three timeswith 200 μL/well PBS, 0.05% Tween (PBST, Sigma). 100 μL/well of adilution series in PBS (Sigma) of the respective antibody containingcell culture supernatants was added to the wells and incubated for 1-2hour on a microtiterplate shaker at room temperature. The wells werewashed three times with 200 μL/well PBST and bound antibody was detectedwith 100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL as the detectionantibody for 1-2 hours on a microtiterplate shaker at room temperature.Unbound detection antibody was washed away three times with 200 μL/wellPBST and the bound detection antibody was detected by addition of 100 μLABTS/well. Determination of absorbance was performed on a Tecan FluorSpectrometer at a measurement wavelength of 405 nm (reference wavelength492 nm).

Protein Purification

Proteins were purified from filtered cell culture supernatants referringto standard protocols. In brief, antibodies were applied to a Protein ASepharose column (GE healthcare) and washed with PBS. Elution ofantibodies was achieved at pH 2.8 followed by immediate neutralizationof the sample. Aggregated protein was separated from monomericantibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20 mM Histidine, 150 mM NaCl pH 6.0. Monomericantibody fractions were pooled, concentrated (if required) using e.g., aMILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, frozen andstored at −20° C. or −80° C. Part of the samples were provided forsubsequent protein analytics and analytical characterization e.g. bySDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry.

SDS-PAGE

The NuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instruction. In particular, 10% or 4-12% NuPAGE® Novex®Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE® MES (reduced gels, withNuPAGE® Antioxidant running buffer additive) or MOPS (non-reduced gels)running buffer was used.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed by HPLCchromatography. Briefly, Protein A purified antibodies were applied to aTosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH₂PO₄/K₂HPO₄, pH 7.5on an Agilent HPLC 1100 system or to a Superdex 200 column (GEHealthcare) in 2×PBS on a Dionex HPLC-System. The eluted protein wasquantified by UV absorbance and integration of peak areas. BioRad GelFiltration Standard 151-1901 served as a standard.

Mass Spectrometry

This section describes the characterization of the multispecificantibodies with VH/VL exchange (VH/VL CrossMabs) with emphasis on theircorrect assembly. The expected primary structures were analyzed byelectrospray ionization mass spectrometry (ESI-MS) of the deglycosylatedintact CrossMabs and deglycosylated/plasmin digested or alternativelydeglycosylated/limited LysC digested CrossMabs.

The VH/VL CrossMabs were deglycosylated with N-Glycosidase F in aphosphate or Tris buffer at 37° C. for up to 17 h at a proteinconcentration of 1 mg/ml. The plasmin or limited LysC (Roche) digestionswere performed with 100 μg deglycosylated VH/VL CrossMabs in a Trisbuffer pH 8 at room temperature for 120 hours and at 37° C. for 40 min,respectively. Prior to mass spectrometry the samples were desalted viaHPLC on a Sephadex G25 column (GE Healthcare). The total mass wasdetermined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik)equipped with a TriVersa NanoMate source (Advion).

Determination of Binding and Binding Affinity of MultispecificAntibodies to the Respective Antigens Using Surface Plasmon Resonance(SPR) (BIACORE)

Binding of the generated antibodies to the respective antigens isinvestigated by surface plasmon resonance using a BIACORE instrument (GEHealthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinitymeasurements Goat-Anti-Human IgG, JIR 109-005-098 antibodies areimmobilized on a CM5 chip via amine coupling for presentation of theantibodies against the respective antigen. Binding is measured in HBSbuffer (HBS-P (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, ph 7.4), 25°C. (or alternatively at 37° C.). Antigen (R&D Systems or in housepurified) was added in various concentrations in solution. Associationwas measured by an antigen injection of 80 seconds to 3 minutes;dissociation was measured by washing the chip surface with HBS bufferfor 3-10 minutes and a KD value was estimated using a 1:1 Langmuirbinding model. Negative control data (e.g. buffer curves) are subtractedfrom sample curves for correction of system intrinsic baseline drift andfor noise signal reduction. The respective Biacore Evaluation Softwareis used for analysis of sensorgrams and for calculation of affinitydata.

TIM3 Antibodies Example 1a: Generation of Anti-TIM3 AntibodiesImmunization of Mice

NMRI mice were immunized genetically, using a plasmid expression vectorcoding for full-length human Tim-3 by intradermal application of 100 ugvector DNA (plasmid 15304_hTIM3-fl), followed by Electroporation (2square pulses of 1000 V/cm, duration 0.1 ms, interval 0.125 s; followedby 4 square pulses of 287.5 V/cm, duration 10 ms, interval 0.125 s. Micereceived either 6 consecutive immunizations at days 0, 14, 28, 42, 56,70, and 84. Blood was taken at days 36, 78 and 92 and serum prepared,which was used for titer determination by ELISA (see below). Animalswith highest titers were selected for boosting at day 96, by intravenousinjection of 50 ug of recombinant human Tim-3 human Fc chimera, andmonoclonal antibodies were isolated by hybridoma technology, by fusionof splenocytes to myeloma cell line 3 days after boost.

Determination of Serum Titers (ELISA)

Human recombinant Tim-3 human Fc chimera was immobilized on a 96-wellNUNC Maxisorp plate at 0.3 ug/ml, 100 μl/well, in PBS, followed by:blocking of the plate with 2% Crotein C in PBS, 200 μl/well; applicationof serial dilutions of antisera, in duplicates, in 0.5% Crotein C inPBS, 100 μl/well; detection with HRP-conjugated goat anti-mouse antibody(Jackson Immunoresearch/Dianova 115-036-071; 1/16 000). For all steps,plates were incubated for 1 h at 37° C. Between all steps, plates werewashed 3 times with 0.05% Tween 20 in PBS. Signal was developed byaddition of BM Blue POD Substrate soluble (Roche), 100 ul/well; andstopped by addition of 1 M HCl, 100 ul/well. Absorbance was read out at450 nm, against 690 nm as reference. Titer was defined as dilution ofantisera resulting in half-maximal signal.

Example 1b: Characterization Anti-TIM3 Antibodies ELISA for TIM3

Nunc-Maxi Sorp Streptavidine plates (MicroCoat #11974998/MC1099) werecoated by 25 μl/well with Tim3-ECD-His-Biotin (biotinylated with BirALigase) and incubated at RT for 1 h while shaking at 400 rpm rotation.After washing (3×90 μl/well with PBST-buffer) 25 μl aTim3 samples ordiluted (1:2 steps) reference antibody aTim3 F38-2E2 (Biolegend) wasadded and incubated 1 h at RT. After washing (3×90 μl/well withPBST-buffer) 25 μl/well sheep-anti-mouse-POD (GE NA9310V) was added in1:9000 dilution and incubated at RT for 1 h while shaking at 400 rpmrotation. After washing (4×90 μl/well with PBST-buffer) 25 μl/well TMBsubstrate (Calbiochem, #CL07) was added and incubated until OD 1.5-2.5.Then the reaction was stopped by addition of 25 μl/well 1N HCL-solution.Measurement took place at 370/492 nm.

ELISA results are listed as EC₅₀-values [ng/ml] in Summary Table 1below.

Cell ELISA for TIM3

Adherent CHO-K1 cell line stably transfected with plasmid15312_hTIM3-fl_pUC_Neo coding for full-length human Tim3 and selectionwith G418 (Neomycin resistance marker on plasmid) were seeded at aconcentration of 1.2×10E6 cells/ml into 384-well flat bottom plates andgrown over night.

At the next day 25 μl/well Tim3 sample or aTim3 reference antibodyF38-2E2 Azide free (Biolegend, 354004) was added and incubated for 2 hat 4° C. (to avoid internalization). After washing (3×90 μl/well PBST(BIOTEK Washer: Prog. 29, 1×90) cells were fixed by flicking outresidual buffer and addition of 50 μl/well 0.05% Glutaraldehyde:Dilution 1:500 of 25% Glutaraldehyde (Sigma Cat. No: G5882) in1×PBS-buffer and incubated for 1 h at RT. After washing (3×90 μl/wellPBST (BIOTEK Washer: Prog. 21, 3×90 GreinLysin) 25 μl/well secondaryantibody was added for detection (Sheep-anti-mouse-POD; Horseradish PODlinked F(ab′)₂ Fragment; GE NA9310) followed by 2 h incubation at RTwhile shaking at 400 rpm. After washing (3×90 μl/well PBST (BIOTEKWasher: Prog. 21, 3×90 GreinLysin) 25 μl/well TMB substrate solution(Roche 11835033001) was added and incubated until OD 1.5-2.5. Then thereaction was stopped by addition of 25 μl/well 1N HCL-solution.Measurement took place at 370/492 nm.

Cell ELISA results are listed as “EC₅₀ CHO-Tim3”-values [ng/ml] insummary Table 1 below.

TABLE 1 Binding affinities of exemplary antibodies (ELISA and BIACORE)Tim3_ Tim3_ Tim3_ Tim3_ Tim3_ Tim3_ Assay 0018 0021 0028 0026 0033 0038Affinity KD [nM] monomer/dimer Tim3 3.4/1.1 204/4.1 173/2.8 6.2/1.5n.f./3.1 7.6/0.6 EC₅₀ ELISA [nM] 0.56 0.22 0.501 EC₅₀ ELISA [ng/ml] 9447 37 47 1321 83 EC₅₀ CHO-Tim3 [nM] 0.52 0.32 0.17 EC₅₀ CHO-Tim3 [ng/ml]87 73 53 69 3710 29

BIAcore Characterization of the TIM3 Antibodies

A surface plasmon resonance (SPR) based assay has been used to determinethe kinetic parameters of the binding between several murine Tim3binders as well as commercial human Tim3 binding references. Therefore,an anti-mouse IgG was immobilized by amine coupling to the surface of a(BIAcore) CM5 sensor chip. The samples were then captured and monomerichu/cy Tim3-ECD as well as a Fc-tagged human Tim3-ECD dimer was bound tothem. The sensor chip surface was regenerated after each analysis cycle.The equilibrium constant K_(D) was finally gained by fitting the data toa 1:1 Langmuir interaction model.

About 12000 response units (RU) of 30 μg/ml anti-mouse IgG (GEHealthcare #BR-1008-38) were coupled onto the spots 1, 2, 4 and 5 of theflow cells 1-4 (spots 1,5 are active and spots 2,4 are reference spots)of a CM5 sensor chip in a BIAcore B4000 at pH 5.0 by using an aminecoupling kit supplied by GE Healthcare.

The sample and running buffer was HBS-EP+ (0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.05% v/v Surfactant P20, pH 7.4). Flow cell temperature wasset to 25° C. and sample compartment temperature to 12° C. The systemwas primed with running buffer.

The samples were injected for 30 seconds with a concentration of 200μg/ml and bound to the spots 1 and 5 of each flow cell, allowing themeasurement of eight samples in parallel. Then a complete set ofdifferent (monomeric cyno, monomeric human and huFc fused dimeric humanTim3-ECD) concentrations was injected over each sample for 240 sfollowed by a dissociation time of 30/1800 s. Each analysis cycle(sample capture, spot 1 and 5—Tim3 ECD injection) was then regeneratedwith a 30 seconds long injection of Glycine-HCl pH 1.7. The flow ratewas set to 30 μl/min for the whole run.

Finally the double referenced data was fitted to a 1:1 Langmuirinteraction model with the BIAcore B4000 Evaluation Software. Resultingaffinities to monomeric human, cyno Tim3 and huFc fused dimeric humanTim3 are shown in Table 2a. The affinity to the hu Tim3 dimer is mostlikely affected by avidity and therefore apparently stronger than theaffinity to the monomeric huTim3.

TABLE 2a Binding affinities determined by BIAcore-KD values gained by akinetic SPR measurement.-n.f. means no fit possible, most likely due tono or weak binding. huTim3 K_(D) huTim3Fc K_(D) cyTim3 K_(D) Sample (25°C.) [M] (25° C.) [M] (25° C.) [M] TIM3-0016 3.29E−09 1.09E−09 2.16E−08TIM3-0016 variant (0018) 3.40E−09 1.11E−09 4.19E−08 TIM3-0021 2.04E−074.07E−09 n.f. TIM3-0022 1.26E−07 1.52E−09 2.84E−08 TIM3-0026 6.23E−091.52E−09 n.f. TIM3-0028 1.73E−07 2.77E−09 n.f. TIM3-0030 3.11E−091.28E−09 n.f. TIM3-0033 n.f. 3.05E−09 n.f. TIM3-0038 7.56E−09 5.69E−10n.f. Reference antibody 1.36E−08 7.50E−09 1.68E−07 Biolegend F38-2E2Reference antibody 1.34E−08 7.73E−09 1.41E−07 USB 11E365

Determination of the Affinity to Tim3 Via SPR (Chimeric TIM3-0016Variant (0018) and Humanized Versions)

Protein A was immobilized by amine coupling to the surface of a(Biacore) CM5 sensor chip. The samples were then captured and huTim3-ECD was bound to them. The sensor chip surface was regeneratedafter each analysis cycle. The equilibrium constant and kinetic rateconstants were finally gained by fitting the data to a 1:1 Langmuirinteraction model.

About 2000 response units (RU) of 20 μg/ml Protein A were coupled ontothe spots 1, 2, 4 and of all flow cells of a CM5 sensor chip in aBiacore B4000 instrument using an amine coupling kit supplied by GEHealthcare.

The sample and running buffer was HBS-EP+(0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.05% v/v Surfactant P20, pH 7.4). Flow cell temperature wasset to 25° C. and sample compartment temperature to 12° C. The systemwas primed with running buffer.

Different samples were injected for 30 seconds with a concentration of10 nM and bound consecutively to the spots 1 and 5 in all flow cells.Then a complete set of monomeric human Tim3-ECD dilutions (600 nM, 200nM, 66.7 nM, 2×22.2 nM, 7.4 nM, 2.5 nM and 2×0 nM) was consecutivelyinjected over each sample for 300 s. Each antigen injection was followedby a dissociation time of 12 s/1000 s and two 30 s regeneration stepswith a Glycine-HCl pH 1.5 solution, of which the last one contained astabilization period after injection of 20 seconds.

Finally the double referenced data was fitted to a 1:1 Langmuirinteraction model using the Biacore B4000 Evaluation Software. ResultingK_(D) values are shown in Table 2b.

Determination of the Affinity to Tim3 Via SPR ((Chimeric TIM3-0028 andHumanized Versions))

Anti-human Fc IgG was immobilized by amine coupling to the surface of a(Biacore) CM5 sensor chip. The samples were then captured and huTim3-ECD was bound to them. The sensor chip surface was regeneratedafter each analysis cycle. The equilibrium constant and kinetic rateconstants were finally gained by fitting the data to a 1:1 Langmuirinteraction model.

About 2500 response units (RU) of 10 μg/ml anti-human Fc IgG (GEHealthcare #BR-1008-39) were coupled onto the spots 1, 2, 4 and 5 of allflow cells of a CM5 sensor chip in a Biacore B4000 instrument using anamine coupling kit supplied by GE Healthcare.

The sample and running buffer was HBS-EP+(0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.05% v/v Surfactant P20, pH 7.4). Flow cell temperature wasset to 25° C. and sample compartment temperature to 12° C. The systemwas primed with running buffer.

Different samples were injected for 30 seconds with a concentration of10 nM and bound consecutively to the spots 1 and 5 in all flow cells.Then a complete set of monomeric human Tim3-ECD dilutions (600 nM, 200nM, 66.7 nM, 2×22.2 nM, 7.4 nM, 2.5 nM and 2×0 nM) was consecutivelyinjected over each sample for 300 s. Each antigen injection was followedby a dissociation time of 12 s/700 s and two 30 s regeneration stepswith a 3 M MgCl₂ solution, of which the last one contained an “extrawash after injection” with running buffer.

Finally the double referenced data was fitted to a 1:1 langmuirinteraction model using the Biacore B4000 Evaluation Software. ResultingK_(D) values are shown in Table 2b.

TABLE 2b Binding affinities determined by BIAcore-KD values gained by akinetic SPR measurement Sample huTim3 K_(D) (25° C.) [M] ChimericTIM3-0016 variant (0018) 2.78E−09 TIM3-0433 5.74E−09 TIM3-0434 5.76E−09Chimeric TIM3-0028 2.35E−07 TIM3-0438 3.05E−07 TIM3-0443 2.87E−07

Example 2: Generation of Anti-TIM3 Antibody Derivatives ChimericAntibody Derivatives

Chimeric Tim3 antibodies were generated by amplifying the variable heavyand light chain regions of the anti-TIM3 mouse antibodies Tim3-0016,Tim3-0016 variant (0018), Tim3-0021, Tim3-0022, Tim3-0026, Tim3-0028,Tim3-0030, and Tim3-0033, Tim3-0038 from via PCR and cloning them intoheavy chain expression vectors as fusion proteins with human IgG1backbones/human CH1-Hinge-CH2-CH3 with LALA and PG mutations (Leucine234 to Alanine, Leucine 235 to Alanine, Proline 329 to Glycine)abrogating effector functions and light chain expression vectors asfusion proteins to human C-kappa. LC and HC Plasmids were thencotransfected into HEK293 and purified after 7 days from supernatants bystandard methods for antibody purification.

Removal of Glycosylation Site NYT: Modifying 1 HVR-L1 Position inTim3-0016, Tim3_0016 Variant (Named 0018 or Tim3_0018) by Substitutionof N by Q or S

Mutations within the variable light chain region of Tim3_0016 andTim3_0016 variant (0018) were generated by in vitro mutagenesis usingAgilent “Quick Change Lightning Site-directed Mutagenesis Kit” accordingmanufacturer's instructions. By this method the asparagine (N) of theglycosylation site motif NYT in the light chain HVR-L1 (SEQ ID NO: 4)was replaced by glutamine (Q) (resulting in SEQ ID NO:11=Tim3_0016_HVR-L1 variant 1 NQ) or, alternatively, the asparagine (N)was replaced by serine (S) (resulting in SEQ ID NO: 12=Tim3_0016_HVR-L1variant 2_NS). In both, the glycosylation site motif NYT wassuccessfully modified. LC and HC Plasmids coding for the variants werethen cotransfected into HEK293 and purified after 7 days fromsupernatants by standard methods for antibody purification.

The generated mutants were tested by ELISA on human Tim3, ELISA oncynomolgus Tim3 and cellular ELISA on adherent CHO-K1 cells expressingfull-length human Tim3.

TABLE 3 Cellular binding Biochem Human Biochem Cyno CHO-TIM3 EC₅₀ EC₅₀EC₅₀ [ng/ml] [ng/ml] [ng/ml] values in values in values in Antibodiesand relation to Inflexion relation to Inflexion relation to Inflexionmutant antibodies the samples point the samples point the samples pointtested max value [ng/ml] max value [ng/ml] max value [ng/ml] Anti Tim3F38-2E2 73.2 88.3 423.0 209871.3 150.2 224.3 Tim3_0018 15.1 15.3 14.614.6 26.4 29.4 (TIM3-0016 variant) Tim3_0018MutNQ 12.0 10.8 13.2 10.813.4 12.8 Tim3_0018MutNS 10.3 6.5 11.9 6.5 11.2 11.1 Tim3_0016MutNQ 7.65.7 8.3 5.7 6.3 5.4 Tim3_0016MutNS 8.5 5.5 9.7 5.5 9.1 8.5

All mutants generated were found to show even more functional binding tohuman TIM3 (human), cyno TIM3 (cyno) or human TIMR on CHO cells than theparental antibodies Tim3_0016 or the Tim3_0016 antibody variantTim3_0018 respectively.

Humanized Antibody Derivatives Humanization of the VH and VL Domains ofMurine Anti-Tim3-0016 Variant (0018) and Anti-Tim3_0028

Based upon the amino acid sequence of the VH and VL domains of a)anti-Tim3 antibody Tim3_0016 variant (0018) (with the amino acidsequences of the 6 HVRs wherein in the light chain the HVR-L1 variant2_NS (removal of glycosylation site by N to S mutation) was usedhumanized anti-Tim3 antibody variants Tim3-0433 and Tim3-0434 weregenerated and based upon the amino acid sequence of the VH and VLdomains of b) anti-Tim3 antibody Tim3_0028 humanized anti-Tim3 antibodyvariants Tim3-0438 and Tim3-0443 were generated.

The humanized amino acid sequences for heavy and light chain variableregions of were backtranslated in to DNA and the resulting cNDA weresynthesized (GenArt) and then cloned into heavy chain expression vectorsas fusion proteins with human IgG1 backbones/human CH1-Hinge-CH2-CH3with LALA and PG mutations (Leucine 234 to Alanine, Leucine 235 toAlanine, Proline 329 to Glycine) abrogating effector functions or intolight chain expression vectors as fusion proteins to human C-kappa. LCand HC Plasmids were then cotransfected into HEK293 and purified after 7days from supernatants by standard methods for antibody purification.The resulting humanized Tim3-antibodies are named as follows:

TABLE 4 VH and VL sequences of humanized antibodies VH/SEQ ID NO: VL/SEQID NO: Humanized antibodies of Tim3_0016 variant (0018) Tim3-0433 SEQ IDNO: 13 SEQ ID NO: 14 Tim3-0434 SEQ ID NO: 15 SEQ ID NO: 16 Humanizedantibodies of Tim3_0028 Tim3-0438 SEQ ID NO: 25 SEQ ID NO: 26 Tim3-0443SEQ ID NO: 27 SEQ ID NO: 28

TABLE 5 HVR sequences of humanized antibodies HVR-H1, HVR-H2, HVR-L1,HVR-L2, and HVR-H3/ and HVR-L3 SEQ ID NOs: t/SEQ ID NOs: Humanizedantibodies of Tim3_0016 variant (0018) Tim3-0433 SEQ ID NOs: 1, SEQ IDNOs: 12, 2 and 3 5 and 6 Tim3-0434 SEQ ID NOs: 1, SEQ ID NOs: 12, 2 and3 5 and 6 Humanized antibodies of Tim3_0028 Tim3-0438 SEQ ID NOs: 17,SEQ ID NOs: 20, 18 and 19 21 and 22 Tim3-0443 SEQ ID NOs: 17, SEQ IDNOs: 20, 18 and 19 21 and 22

Example 3: Effect of Human Anti-TIM-3 Antibodies on Cytokine Productionin a Mixed Lymphocyte Reaction (MLR)

A mixed lymphocyte reaction was used to demonstrate the effect ofblocking the TIM-3 pathway to lymphocyte effector cells. T cells in theassay were tested for activation and IFN-gamma secretion in the presenceor absence of an anti-TIM-3 mAbs.

Human Lymphocytes were isolated from peripheral blood of healthy donorby density gradient centrifugation using Leukosep (Greiner Bio One, 227288). Briefly, heparinized blood was diluted with the three fold volumeof PBS and 25 ml aliquots of the diluted blood were layered in 50 mlLeukosep tubes. After centrifugation at 800×g for 15 min at roomtemperature (w/o break) the lymphocyte containing fractions wereharvested, washed in PBS and used directly in functional assay orresuspended in freezing medium (10% DMSO, 90% FCS) at 1.0E+07 cells/mland stored in liquid nitrogen. 1:1 target/responder cell ratio was usedin MLR assay (i.e. each MLR culture contained—2.0E+05 PBMCs from eachdonor in a total volume of 200 μl. Anti-TIM3 monoclonal antibodiesTim3_0016, Tim3_0016 variant (Tim3_0018), Tim3_0021, Tim3_0022,Tim3_0026, Tim3_0028, Tim3_0030, Tim3_0033, Tim3_0038 and F38-2E2(BioLegend), were added to each culture at different antibodyconcentrations. Either no antibody or an isotype control antibody wasused as a negative control and rec hu IL-2 (20 EU/ml) was used aspositive control. The cells were cultured for 6 days at 37° C. After day6 100 μl of medium was taken from each culture for cytokine measurement.The levels of IFN-gamma were measured using OptEIA ELISA kit (BDBiosciences).

The results are shown in Table 6 (IFN-g secretion/release). Theanti-TIM-3 monoclonal antibodies promoted T cell activation andIFN-gamma secretion in concentration dependent manner. The anti-TIM3antibodies Tim3_0021, Tim3 0022, Tim3_0028, and Tim3_0038 reduce releaseof the inflammatory cytokine IFN-gamma) more than the F38-2E2 antibody.Tim3_0016, Tim3_0016 variant (Tim3_0018), Tim3_0033 and Tim3_0038 showeda similar release when compared the F38-2E2 antibody. In contrast,cultures containing the isotype control antibody did not show anincrease in IFN-gamma secretion.

TABLE 6a Percentage of anti-Tim3 antibody induced IFNgamma release incomparison to rec hu IL-2 (20 EU/ml) (=100%) as positive control and noantibody as negative control MLR + Iso- Iso- Compound IL-2 type F38-Tim3 Tim3 Tim3 Tim3 Tim3 Tim3 Tim3 Tim3 Tim3 type concentration 20 U/mlIgG2a 2E2 0016 0018 0021 0022 0026 0028 0030 0033 0038 hIgG1 40 μg/ml 236 33 36 112 58 25 40 14 35 51 0 10 μg/ml 100 0 26 22 30 108 38 16 38 430 38 5  1 μg/ml 0 7 7 12 101 18 18 12 3 0 1 0

In further experiments the EC50 values of the following chimeric andhumanized antibodies (generated as described above) in combination with0.1 μg/ml anti-PD1 mAb were measured: chimeric chi_Tim3_018 antibody andits humanized versions Tim3-433 and Tim3-434, chimeric chi_Tim3_028antibody and its humanized versions Tim3-438 and Tim3-443 were measuredwith different lymphocyte donor mixtures (D2 and D3, or D1 and D5,respectively) Results are shown in Table 6b.

TABLE 6b EC₅₀ of anti-Tim3 antibody induced (IFN-g secretion/release)EC₅₀ [nM] EC₅₀ [nM] Antibody with donors D2 + D3 with donors D1 + D5chi_Tim3_018 3.1 4.2 Tim3-433 3.0 2.4 Tim3-434 1.7 2.6 chi_Tim3_028 2.96.4 Tim3-438 1.9 2.7 Tim3-443 3.0 4.7

PD1 Antibodies Example 4: Generation of Anti-PD-1 AntibodiesImmunization of Mice

NMRI mice were immunized genetically, using a plasmid expression vectorcoding for full-length human PD-1 by intradermal application of 100 ugvector DNA (plasmid15300_hPD1-fl), followed by Electroporation (2 squarepulses of 1000 V/cm, duration 0.1 ms, interval 0.125 s; followed by 4square pulses of 287.5 V/cm, duration 10 ms, interval 0.125 s. Micereceived either 6 consecutive immunizations at days 0, 14, 28, 42, 56,70, and 84. Blood was taken at days 36, 78 and 92 and serum prepared,which was used for titer determination by ELISA (see below). Animalswith highest titers were selected for boosting at day 96, by intravenousinjection of 50 ug of recombinant human PD1 human Fc chimera, andmonoclonal antibodies were isolated by hybridoma technology, by fusionof splenocytes to myeloma cell line 3 days after boost.

Determination of Serum Titers (ELISA)

Human recombinant PD1 human Fc chimera was immobilized on a 96-well NUNCMaxisorp plate at 0.3 ug/ml, 100 ul/well, in PBS, followed by: blockingof the plate with 2% Crotein C in PBS, 200 ul/well; application ofserial dilutions of antisera, in duplicates, in 0.5% Crotein C in PBS,100 ul/well; detection with HRP-conjugated goat anti-mouse antibody(Jackson Immunoresearch/Dianova 115-036-071; 1/16 000). For all steps,plates were incubated for 1 h at 37° C. Between all steps, plates werewashed 3 times with 0.05% Tween 20 in PBS. Signal was developed byaddition of BM Blue POD Substrate soluble (Roche), 100 ul/well; andstopped by addition of 1 M HCl, 100 ul/well. Absorbance was read out at450 nm, against 690 nm as reference. Titer was defined as dilution ofantisera resulting in half-maximal signal.

Example 5: Characterization Anti-PD1 Antibodies/Binding of Anti-PD1Antibodies to Human PD1 ELISA for hu PD1

Nunc maxisorp streptavidin coated plates (MicroCoat #11974998001) werecoated with 25 μl/well biotinylated PD1-ECD-AviHis and incubated at 4°C. over night. After washing (3×90 μl/well with PBST-buffer) 25 μl antiPD1 samples or reference antibodies (human anti PD1; Roche/mouse antiPD1; Biolegend; cat.:329912) were added and incubated 1 h at RT. Afterwashing (3×90 μl/well with PBST-buffer) 25 μl/well goat-anti-humanH+L-POD (JIR, JIR109-036-088)/Sheep-anti-mouse-POD (GE Healthcare;NA9310) was added in 1:2000/1:1000 dilution and incubated at RT for 1 hon shaker. After washing (3×90 μl/well with PBST-buffer) 25 μl/well TMBsubstrate (Roche Catalogue No. 11835033001) was added and incubateduntil OD 2-3. Measurement took place at 370/492 nm.

ELISA results are listed as EC50-values [ng/ml] in summary Tables 7 and8 below.

Cell ELISA for PD1

Adherent CHO-K1 cell line stably transfected with plasmid15311_hPD1-fl_pUC_Neo coding for full-length human PD1 and selectionwith G418 (Neomycin resistance marker on plasmid) were seeded at aconcentration of 0.01×10E6 cells/well in 384-well flat bottom plates andgrown over night.

The next day 25 μl/well PD1 sample or human anti PD1 (Roche)/mouse antiPD1(Biolegend; cat.:329912) reference antibody were added and incubatedfor 2 h at 4° C. (to avoid internalization). After washing carefully(1×90 μl/well PBST) cells were fixed by adding 30 μl/well 0.05%Glutaraldehyde (Sigma, Cat. No: G5882, 25%) diluted in 1×PBS-buffer andincubated for 10 min at RT. After washing (3×90 μl/well PBST) 25 μl/wellsecondary antibody was added for detection: goat-anti-human H+L-POD(JIR, JIR109-036-088)/Sheep-anti-mouse-POD (GE NA9310) followed by 1 hincubation at RT on shaker. After washing (3×90 μl/well PBST) 25 μl/wellTMB substrate solution (Roche 11835033001) was added and incubated untilOD 1.0-2.0. Plates were measured at 370/492 nm.

Cell ELISA results are listed as “EC50 CHO-PD1”-values [ng/ml] insummary table Table 8 below.

ELISA for Cyno PD1

Nunc maxisorp streptavidin coated plates (MicroCoat #11974998001) werecoated with 25 μl/well biotinylated cynoPD1-ECD-Biotin and incubated at4° C. over night. After washing (3×90 μl/well with PBST-buffer) 25 μlanti PD1 samples or reference antibodies (human anti PD1; Roche) wereadded and incubated 1 h at RT on shaker. After washing (3×90 μl/wellwith PBST-buffer) 25 μl/well goat-anti-human H+L-POD (JIR,JIR109-036-088) was added in 1:1000 dilution and incubated at RT for 1 hon shaker. After washing (3×90 μl/well with PBST-buffer) 25 μl/well TMBsubstrate (Roche, 11835033001) was added and incubated until OD 2-3.Measurement took place at 370/492 nm.

ELISA results are listed as EC50-values [ng/ml] in summary Table 7 and 8below.

PD Ligand 1 Replacing Assay

Nunc maxisorp streptavidin coated plates (MicroCoat #11974998001) werecoated with 25 μl/well biotinylated PD1-ECD-AviHis and incubated at 4°C. over night. After washing (3×90 μl/well with PBST-buffer) 25 μl antiPD1 samples or reference antibodies (mouse anti PD1; Biolegend;cat.:329912) were added and incubated 1 h at RT on shaker. After washing(3×90 μl/well with PBST-buffer) 25 μl/well PD-L1 (Recombinant humanB7-H1/PD-L1 Fc Chimera; 156-B7, R&D) was added and incubated 1 h at RTon shaker. After washing (3×90 μl/well with PBST-buffer) 25 μl/wellgoat-anti-human H+L-POD (JIR, 109-036-088) was added in 1:1000 dilutionand incubated at RT for 1 h on shaker. After washing (3×90 μl/well withPBST-buffer) 25 μl/well TMB substrate (Roche, 11835033001) was added andincubated until OD 2-3. Measurement took place at 370/492 nm.

ELISA results are listed as IC₅₀-values [ng/ml] in summary Table 7below.

PD Ligand 2 Replacing Assay

Nunc maxisorp streptavidin coated plates (MicroCoat #11974998001) werecoated with 25 μl/well biotinylated PD1-ECD-AviHis and incubated at 4°C. over night. After washing (3×90 μl/well with PBST-buffer) 25 μl antiPD1 samples or reference antibodies (mouse anti huPD1; Roche) were addedand incubated 1 h at RT on shaker. After washing (3×90 μl/well withPBST-buffer) 25 μl/well PD-L2 (Recombinant human B7-DC/PD-L2 Fc Chimera;1224-PL-100, R&D) was added and incubated 1 h at RT on shaker. Afterwashing (3×90 μl/well with PBST-buffer) 25 μl/well goat-anti-humanH+L-POD (JIR, 109-036-088) was added in 1:2000 dilution and incubated atRT for 1 h on shaker. After washing (3×90 μl/well with PBST-buffer) 25μl/well TMB substrate (Roche, 11835033001) was added and incubated untilOD 2-3. Measurement took place at 370/492 nm.

ELISA results are listed as IC₅₀-values [ng/ml] in summary Table 7below.

Epitope Mapping ELISA/Binding Competition Assay

Nunc maxisorp plates (Nunc #464718) were coated with 25 μl/well captureantibody (goat anti mouse IgG; JIR; 115-006-071) and incubated for 1 hat RT on shaker. After washing (3×90 μl/well with PBST-buffer) plateswere blocked for 1 h with 2% BSA containing PBS buffer at RT on shaker.After washing (3×90 μl/well with PBST-buffer) 25 μl mouse anti PD1samples were added and incubated 1 h at RT on shaker. After washing(3×90 μl/well with PBST-buffer) capture antibody was blocked by 30μl/well mouse IgG (JIR; 015-000-003) for 1 h at RT on shaker. At thesame time biotinylated PD1-ECD-AviHis was preincubated with secondsample antibody for 1 h at RT on shaker. After washing assay plate (3×90μl/well with PBST-buffer) the PD1 antibody mix was transferred to assayplate and incubated at RT for 1 h on shaker. After washing (3×90 μl/wellwith PBST-buffer) 25 μl/well streptavidin POD (Roche, #11089153001) wasadded in 1:4000 dilution and incubated at RT for 1 h on shaker. Afterwashing (3×90 μl/well with PBST-buffer) 25 μl/well TMB substrate (Roche,#11089153001) was added and incubated until OD 1.5-2.5. Measurement tookplace at 370/492 nm. Epitope groups were defined by hierarchicalclustering against reference antibodies.

TABLE 7 Binding, PD-L1 inhibition and epitope region groups of exemplaryantibodies (ELISA) Epitope ELISA ELISA region ELISA ELISA PD-L1 PD-L2group huPD1 cyPD1 inhibition inhibition By EC₅₀ EC₅₀ IC₅₀ IC₅₀ competionAntibody [ng/ml] [ng/ml] [ng/ml] [ng/ml] assay) PD1-0050 17.9 9.8 128 341 PD1-0069 45.7 22.7 225 89 6 PD1-0073 15.1 8.3 124 65 5 PD1-0078 26.322.4 x 86 2 PD1-0098 50.8 54.6 174 45 5 PD1-0102 34.2 52.7 >35.5 μg/ml140 4 PD1-0103 33.7 36.9 182 51 5

TABLE 8 Biochemial-and Cell-binding of humanized PD1 antibodies derivedfrom parental mouse antibody PD1-0103 (ELISA). ELISA Humanized huPD1ELISA cyPD1 ELISA CHO-PD1 antibody EC₅₀ [ng/ml] EC₅₀ [ng/ml] EC₅₀[ng/ml] PD1-103- 11 8.3 10.1 0312 PD1-103- 15 11 10.8 0313 PD1-103- 118.3 7.7 0314 PD1-103- 10 7.9 7.3 0315

Biacore characterization of the humanized anti-PD-1 antibodies A surfaceplasmon resonance (SPR) based assay has been used to determine thekinetic parameters of the binding between several murine PD1 binders aswell as commercial human PD1 binding references. Therefore, ananti-human IgG was immobilized by amine coupling to the surface of a(Biacore) CM5 sensor chip. The samples were then captured and hu PD1-ECDwas bound to them. The sensor chip surface was regenerated after eachanalysis cycle. The equilibrium constant and kinetic rate constants werefinally gained by fitting the data to a 1:1 langmuir interaction model.

About 2000 response units (RU) of 20 μg/ml anti-human IgG (GE Healthcare#BR-1008-39) were coupled onto the flow cells 1 and 2 (alternatively: 3and 4) of a CM5 sensor chip in a Biacore T200 at pH 5.0 by using anamine coupling kit supplied by GE Healthcare.

The sample and running buffer was HBS-EP+(0.01 M HEPES, 0.15 M NaCl, 3mM EDTA, 0.05% v/v Surfactant P20, pH 7.4). Flow cell temperature wasset to 25° C. and sample compartment temperature to 12° C. The systemwas primed with running buffer.

The samples were injected for 20 seconds with a concentration of 10 nMand bound to the second flow cell. Then a complete set of human PD1-ECDconcentrations (144 nM, 48 nM, 16 nM, 5.33 nM, 1.78 nM, 0.59 nM, 0.20 nMand 0 nM) was injected over each sample for 120 s followed by adissociation time of 30/300 s and two 20 s regeneration steps with 3 MMgCl₂, of which the last one contained an “extra wash after injection”with running buffer.

Finally the double referenced data was fitted to a 1:1 langmuirinteraction model with the Biacore T200 Evaluation Software. ResultingK_(D), k_(a) and k_(d) values are shown in Table 9.

TABLE 9 Kinetic rate constants and equilibrium constant for chimericPD1-0103 and humanized PD1-Abs determined by Biacore. Ligand ka [M⁻¹s⁻¹]k_(d) [s⁻¹] K_(D) [nM] chimeric PD1-0103 3.86E+05 3.07E−04 0.8PD1-0103-0312 1.95E+05 3.45E−04 1.8 PD1-0103-0313 1.60E+05 3.67E−04 2.3PD1-0103-0314 1.87E+05 2.79E−04 1.5 PD1-0103-0315 1.89E+05 2.91E−04 1.5

As shown in Table 9, all the humanized versions of chimeric PD1-0103(generation see Example 6) display kinetic properties similar to theparental antibody (chimeric PD1-0103).

Kinetics

A CM5 sensor series S was mounted into the Biacore 4000 System and thedetection spots were hydrodynamically addressed according to themanufacturer's instructions.

The polyclonal rabbit IgG antibody <IgGFCγM>R (Jackson ImmunoResearchLaboratories Inc.) was immobilized at 10 000 Ru on the detection spots 1and 5 in the flow cells 1, 2, 3 and 4. Coupling was done via EDC/NHSchemistry according to the manufacturer's instructions. The remainingspots in the flow cells served as a reference. The sample buffer was thesystem buffer supplemented with 1 mg/ml carboxymethyldextrane.

In one embodiment the assay was driven at 25° C. In another embodimentthe assay was driven at 37° C. 50 nM of each murine monoclonal antibodywas captured on the sensor surface by a 1 min injection at 10 μl/min.Subsequently the respective antigens were injected in a concentrationseries of 100 nM, 2×33 nM, 11 nM, 4 nM, 1 nM and system buffer 0 nM at30 μl/min for 4 min association phase time. The dissociation wasmonitored for another 4 min. The capture system was regenerated using a3 min injection of 10 mM glycine pH 1.5 at 30 μl/min. Relevant kineticdata was calculated using the Biacore evaluation software according tothe manufacturer's instructions.

Epitope Mapping

A Biacore 4000 instrument was mounted with a Biacore CAP sensor and wasprepared like recommended by the manufacturer. The instrument buffer wasHBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/v Tween20). The instrument was running at 25° C.

All samples were diluted in system buffer. A 35 kDa biotinylated antigenPD1-ECD-AviHis was captured at 200 RU on the CAP sensor surface by a 1min injection at 30 μl/min in the flow cells 1, 2, 3 and 4 in the spots1 and 5. Spots 2, 3 and 4 served as a reference. In another embodiment,a 35 kDa biotinylated antigen PD1-ECD-AviHis was captured at 200 RU onthe CAP sensor in the same manner.

Subsequently a primary antibody was injected at 100 nM for 3 min at 30μl/min followed by the injection of a secondary antibody at 100 nM for 3min at 30 μl/min. The primary antibody was injected until fullsaturation of the surface presented antigen. At the end of the primaryand secondary antibody injection phases report points “Binding Late”(BL) were set to monitor the binding response of the respectiveantibodies. The Molar Ratio, a quotient between the secondary antibodybinding response “BL2” and the primary antibody response “BL1” wascalculated. The Molar Ratio was used as an indicator of the antigenaccessibility of the secondary antibody, when the antigen was alreadycomplexed by the primary antibody.

The complexes were completely removed from the sensor surface by aninjection for 2 min at 30 μl/min 2M guanidine-HCL 250 mM NaOHregeneration buffer as recommended by the manufacturer, followed by a 1min injection at 30 μl/min of system buffer.

Example 6: Effect of Different Anti-PD-1 Antibodies on CytokineProduction in a Mixed Lymphocyte Reaction (MLR)

3A) The Mixed Lymphocyte Reaction (MLR) is a immune cell assay whichmeasures the activation of lymphocytes from one individual (donor X) tolymphocytes from another individual (donor Y). A mixed lymphocytereaction was used to demonstrate the effect of blocking the PD1 pathwayto lymphocyte effector cells. T cells in the assay were tested foractivation and theier IFN-gamma secretion in the presence or absence ofan anti-PD1 mAbs.

To perform an allogeneic MLR, peripheral blood mononuclear cells (PBMCs)from at least four healthy donors of unknown HLA type were isolated bydensity gradient centrifugation using Leukosep (Greiner Bio One, 227288). Briefly, heparinized blood samples were diluted with the threefold volume of PBS and 25 ml aliquots of the diluted blood were layeredin 50 ml Leukosep tubes. After centrifugation at 800×g for 15 min atroom temperature (w/o break) the lymphocyte containing fractions wereharvested, washed in PBS and used directly in functional assay orresuspended in freezing medium (10% DMSO, 90% FCS) at 1.0E+07 cells/mland stored in liquid nitrogen. Individual 2-way MLR reactions were setup by mixing PBMCs from two different donors at a 1:1stimulator/responder cell ratio and co-cultures were done at least induplicate in flat-bottomed 96-well plates for 6 days at 37° C., 5% CO2,in the presence or w/o of a different concentration range of purifiedanti-PD1 monoclonal antibodies PD1-0050, PD1-0069, PD1-0073, PD1-0078,PD1-0098, PD1-0102, PD1-0103. As reference anti-PD1 antibodies,antibodies comprising the VH and VL domains of either nivolumab (alsoknown as MDX-5C4 or MDX-1106) or pembrolizumab (also known as MK-3475 orOrg 1.09A) were synthesized and cloned with backbones of human IgG1(with mutations L234A, L235A and P329G (EU index of Kabat)). Either noantibody or an isotype control antibody was used as a negative controland rec hu IL-2 (20 EU/ml) was used as positive control. After day 6 100μl of medium was taken from each culture for cytokine measurement. Thelevels of IFN-gamma were measured using OptEIA ELISA kit (BDBiosciences).

The results are shown in Table 10 (IFN-g secretion/release). Theanti-PD1 monoclonal antibodies promoted T cell activation and IFN-gammasecretion in concentration dependent manner. The value of % increase ofIFNg secretion was calculated in relation to IFN-g production of MLR w/oadding of any blocking mAbs (basal allogeneic stimulation induced IFNgvalue as E−c) and MLR with adding of 20 EU/ml rec hu IL-2 (positivecontrol=100% IFNg value as E+c) and was calculated according to formula:Rel. Stimulation [%]=((Esample−E−c)/(E+c−E−c)*100

TABLE 10 Percentage of of IFN gamma secretion after allogenicstimulation and treatment with anti-PD-1 antibody in comparison toeffect of recombinant human IL-2 treatment (20 EU/ml) (= 100% increase)as positive control Concentration Effect (μg/ml) 1:12 1:120 1:1200 inMLR PD1-0050 44 136 96 33 +++ PD1-0069 60 76 71 55 +++ PD1-0073 43 10363 38 ++ PD1-0078 64 99 72 21 ++

Several PD1 blocking antibodies PD1-0050, PD1-0069, PD1-0073, PD1-0078,PD1-0098, PD1-0102, PD1-0103 demonstrated strong immune modulatingactivity by enhancing secretion of interferon gamma (IFN-g) (data notshown for all antibodies).

3B) In a further experiment chimeric PD1-0103 (human IgG1 isotype withmutations L234A, L235A and P329G (EU index of Kabat)) was evaluated.Blockade of PD1 with chimeric PD1-0103 strongly enhances IFN-gammasecretion by allogenic stimulated primary human T cells. ChimericPD1-0103 is more potent than reference anti-PD1 antibodies (see FIG. 1).

For comparison the reference anti-PD1 antibodies comprising the VH andVL domains of either nivolumab (also known as MDX5C4 or MDX-1106) andpembrolizumab (also known as MK-3475 or Org 1.09A) were synthesized andcloned with backbones of human IgG1 (with mutations L234A, L235A andP329G (EU index of Kabat)) were used.

3C) In additional experiments the immune modulating activity of thehumanized variants of anti-PD-1 antibody PD1-0103 (humanized antibodiesPD1-0103-0312, PD1-0103-0314, in FIGS. 2 and 3, see also Example 9below) the a) IFN release (secretion) b) TNF-alpha release (secretion)was evaluated in MLR as described above. The effect of the chimericPD1-0103 antibody and its humanized versions were compared to thereference anti-PD1 antibodies comprising the VH and VL domains of eithernivolumab (also known as MDX5C4 or MDX-1106) and pembrolizumab (alsoknown as MK-3475 or Org 1.09A) with backbones of human IgG1 (withmutations L234A, L235A and P329G (EU index of Kabat)). After 6 days ofMLR culture 50 μl of supernatant was taken and multiple cytokines weremeasured in a single culture using Bio-Plex Pro™ Human Cytokine Th1/Th2Assay (Bio-Rad Laboratories Inc.). (data not shown for all cytokines).

The chimeric PD1-0103 antibody and its humanized versions (PD1-0103_0312and PD1-0103_0314) were more potent compared to the reference anti-PD1antibodies in enhancing the T cell activation and IFN-gamma secretion(see FIG. 2).

Furthermore, the chimeric PD1-0103 antibody and its humanizationvariants increase tumor necrosis factor alpha (TNF alpha) (see FIG. 3)and IL-12 (data not shown) secretion by antigen presenting cells andencance capacity of monocytes/macrophages or antigen presenting cells tostimulate a T cell.

Example 7: Effect of Anti-PD-1 Blockade on Cytotoxic Granzyme B Releaseand IFN-γ Secretion by Human CD4 T Cells Cocultured with AllogeneicMature Dendritic Cells

To further investigate the effect of anti-PD-1 treatment in anallogeneic setting we developed an assay in which freshly purified CD4 Tcells are cocultured for 5 days in presence of monocyte-derivedallogeneic mature dendritic cells (mDCs). Monocytes were isolated fromfresh PBMCs one week before through plastic adherence followed by theremoval of the non-adherent cells. We then generated immature DCs fromthe monocytes by culturing them for 5 days in media containing GM-CSF(50 ng/ml) and IL-4 (100 ng/ml). To induce iDCs maturation, we addedTNF-α, IL-10 and IL-6 (50 ng/ml each) to the culturing media for 2additional days. We then assessed DCs maturation by measuring theirsurface expression of Major Histocompatibility Complex Class II (MHCII),CD80, CD83 and CD86 thorugh flow cytometry (LSRFortessa, BDBiosciences).

On the day of the minimal mixed lymphocyte reaction (mMLR), CD4 T cellswere enriched via a microbead kit (Miltenyi Biotec) from 10⁸ PBMCsobtained from an unrelated donor. Prior culture, CD4 T cells werelabeled with 5 μM of Carboxy-Fluorescein-Succinimidyl Esther (CFSE). 10⁵CD4 T cells were then plated in a 96 well plate together with matureallo-DCs (5:1) in presence or absence of blocking anti-PD1 antibody(either PD1-0103, chimeric PD1-0103, or humanized antibodiesPD1-0103-0312, PD1-0103-0313, PD1-0103-0314, PD1-0103-0315, abbreviatedas 0312, 0313, 0314, 0315 in FIGS. 4A and 4 B), at the concentration of10 μg/ml if not differentely indicated in the figures.

Five days later we collected the cell-culture supernatants, used laterto measure the IFN-γ levels by ELISA (R&D systems), and left the cellsat 37 C degrees for additional 5 hours in presence of Golgi Plug(Brefeldin A) and Golgi Stop (Monensin). The cells were then washed,stained on the surface with anti-human CD4 antibody and the Live/Deadfixable dye Aqua (Invitrogen) before being fixed/permeabilized withFix/Perm Buffer (BD Bioscience). We performed intracellular staining forGranzyme B (BD Bioscience), IFN-γ and IL-2 (both from eBioscience).

We also tested different concentrations of the humanized variantsPD1-0103 (humanized antibodies PD1-0103-0312, PD1-0103-0313,PD1-0103-0314, PD1-0103-0315, abbreviated as 0312, 0313, 0314, 0315 inthe figures, see also Example 9 below) and found them to be equally goodin enhancing granzyme B and interferon gamma. DP47 is a non bindinghuman IgG with a LALA mutation in the Fc portion to avoid recognition byFcγR and was used as negative control. Results are shown in FIGS. 4A and4 B.

Example 8: Chimeric Antibodies Derivatives

Chimeric PD1 antibodies were generated by amplifying the variable heavyand light chain regions of the anti-PD1 mouse antibodies PD1-0098,PD1-0103 via PCR and cloning them into heavy chain expression vectors asfusion proteins with human IgG1 backbones/human CH1-Hinge-CH2-CH3 withmutations L234A, L235A and P329G (EU index of Kabat)) (Leucine 234 toAlanine, Leucine 235 to Alanine, Proline 329 to Glycine) abrogatingeffector functions and light chain expression vectors as fusion proteinsto human C-kappa. LC and HC Plasmids were then cotransfected into HEK293and purified after 7 days from supertnatants by standard methods forantibody purification. The chimeric PD1-antibodies were renamed chimericchiPD1-0098 (chiPD1-0098) and chimeric PD1-0103 (chiPD1-0103). Forcomparison the reference anti-PD1 antibodies comprising the VH and VLdomains of either nivolumab (also known as MDX-5C4 or MDX-1106) andpembrolizumab (also known as MK-3475 or Org 1.09A) were synthesized andcloned with backbones of human IgG1 (with mutations L234A, L235A andP329G (EU index of Kabat)) were used.

Example 9: Generation, Expression and Purification of Humanized Variantsof Anti-PD1 Antibody PD-0103 (huMab PD-0103) and Characterization

Humanization of the VH and VL domains of murine anti-PD1 antibody 0103Based upon the amino acid sequence of the murine VH and VL domains ofmurine anti-PD1 antibody PD1-0103 (SEQ ID NO: 43 and 44), humanizedanti-anti-PD1 antibody variants were generated.

The humanized VH-variant is based on the human germline IMGT_hVH_3_23 incombination with the human J-element germline IGHJ5-01 with severalmutations. (resulting in SEQ ID NO: 45).

The humanized variants of VL are based on the human germlinesIMGT_hVK_4_1, IMGT_hVK_2 30, IMGT_hVK_3_11 and IMGT_hVK_1_39 incombination with the human J-element germline IGKJ1-01. Differentmuations resulted in humanized variants of SEQ ID NO: 46 to SEQ ID NO:49.

The humanized amino acid sequences for heavy and light chain variableregions of PD1-0103 were backtranslated in to DNA and the resulting cNDAwere synthesized (GenArt) and then cloned into heavy chain expressionvectors as fusion proteins with human IgG1 backbones/humanCH1-Hinge-CH2-CH3 with LALA and PG mutations (Leucine 234 to Alanine,Leucine 235 to Alanine, Proline 329 to Glycine) abrogating effectorfunctions or into light chain expression vectors as fusion proteins tohuman C-kappa. LC and HC Plasmids were then cotransfected into HEK293and purified after 7 days from supertnatants by standard methods forantibody purification. The resulting humanized PD1-antibodies named asfollows:

TABLE 11 VH and VL sequences of humanized variant antibodies of PD1-0103Humanized antibodies humanized variant of humanized variant of ofPD1-0103 VH/SEQ ID NO: VL/SEQ ID NO: PD1-0103-0312 SEQ ID NO: 45 SEQ IDNO: 46 PD1-0103-0313 SEQ ID NO: 45 SEQ ID NO: 47 PD1-0103-0314 SEQ IDNO: 45 SEQ ID NO: 48 PD1-0103-0315 SEQ ID NO: 45 SEQ ID NO: 49

TABLE 12 HVR sequences of humanized variant antibodies of PD1-0103HVR-H1, HVR-H2, Humanized and HVR-H3 of HVR-L1, HVR-L2, and antibodieshumanized HVR-L3 of humanized of PD1-0103 variant/SEQ ID NO: variant/SEQID NO: PD-0103-0312 SEQ ID NOs: 37, 38 SEQ ID NOs: 40, 41 and 42 and 39PD-0103-0313 SEQ ID NOs: 37, 38 SEQ ID NOs: 40, 41 and 42 and 39PD-0103-0314 SEQ ID NOs: 37, 38 SEQ ID NOs: 40, 41 and 42 and 39PD-0103-0315 SEQ ID NOs: 37, 38 SEQ ID NOs: 40, 41 and 42 and 39Humanized PD1-0103 antibody variants and parental chimeric PD1-0103 werecharacterized as described above. Results are shown in Table 13.

TABLE 13 Summary of results for humanized PD1-0103 antibody variants andparental chimeric PD1-0103 chimeric PD-0103- PD-0103- PD-0103- PD-0103-Assay PD1-0103 0312 0313 0314 0315 Affinity K_(D 37° C.) 2.0/0.8 1.5/1.81.9/2.3 1.6/1.5 1.7/1.5 [nM] *) ELISA EC₅₀ 0.2 0.1 0.07 0.07 0.06 [nM]CHO-PD1 + + + + + EC₅₀ IC₅₀ PD-L1, 2 1.35 tbd tbd tbd tbd [nM] Mixed ++++++ +++ ++++ ++ Lymphocyte Reaction assay cynomolgus + 0.08 0.06 0.050.04 crossreactivity (EC₅₀ [nm])

Example 10: Neutralizing Potency of PD-1 Antibodies

To test the neutralizing potency of inhouse generated PD-1 antibodies inmimicking a restoration of a suppressed T cell response in vitro acommercially available PD1/PD-L1 reporter assay (Promega) was used. Thissystem consists of PD1+ NFAT Jurkat cells and a PD-L1+ CHO counterpart,which also gives the activation signal. In principle, the reportersystem is based on three steps: (1) TCR-mediated NFAT activation, (2)inhibition of NFAT signal upon activation by the PD-1/PD-L1 axis and (3)recovery of the NFAT signal by PD-1 blocking antibodies.

Material and Methods:

-   -   PD-L1 Medium: PAN Biotech (#P04-03609); FBS (10%) and L-Gln (4        mM)    -   Assay Medium: RPMI 1640 (#31870; Invitrogen), 25 mM HEPES, 2 mM        L-Gln, FBS (2%)    -   Cells used for this assay (both cell types purchased by        Promega):        -   PD-L1+ CHO cells (batch no. #139147): 2-3×104 cells/96 well        -   PD-1+ NFAT Jurkat cells (batch no. #133024: 3.5×104            cells/well

On day 1, PD-L1+ cells were thawed, seeded at the indicated cellconcentration in the above mentioned medium and cultured over night at37° C. and 5% CO₂. On the next day, medium was removed and PD-L1+ cellswere incubated with the prepared antibodies at indicated concentrations(in Assay Medium). In parallel, PD-1+ NFAT Jurkat cells were thawed andabove mentioned cell numbers were transferred to and co-cultured withthe PD-L1+ cells. After an incubation of 6 hrs at 37° C. and 5% CO₂,Bio-Glo substrate was warmed to room temperature (1-2 hrs prioraddition). The cell culture plate was removed from the incubator andadjusted to room temperature (10 min) before 80 μl Bio-Glo solution wasadded per well, incubated for 5-10 min before the luminescence wasmeasured at a Tecan Infinite reader according to the kit'smanufacturer's recommendation. Results can be seen in the FIGS. 5A and 5B where the restoration of a PD-1/PD-L1 mediated suppression of the NFATsignal by different PD-1 antibodies upon TCR stimulation is shown: FIG.5 A: Chimeric PD1_0103 showed a reproducibly superior effect whencompared to a reference antibody. As reference an anti-PD1 antibodycomprising the VH and VL domains nivolumab (also known as MDX-5C4 orMDX-1106) was synthesized and cloned with backbones of human IgG1 (withmutations L234A, L235A and P329G (EU index of Kabat)). FIG. 5B: The fourhumanized variants of PD1_0103 demonstrated a similar in vitro potencyto the lead antibody and were also slightly superior to the referenceantibody.

Bispecific PD1/TIM3 Antibodies Example 11A

Production and Expression of Multispecific Antibodies which Bind to PD1and TIM3 with VH/VL Domain Exchange/Replacement (CrossMAb^(Vh-VL)) inOne Binding Arm and with Single Charged Amino Acid Substitutions in theCH1/CL Interface

In an example multispecific antibodies which binds to human PD1 andhuman TIM3 were generated is described in the general methods section byclassical molecular biology techniques and were expressed transiently in293F of Expi293F cells as described above. The multispecific 1+1CrossMAb^(Vh-Vl) antibodies are described also in WO 2009/080252. Themultispecific antibodies were expressed using expression plasmidscontaining the nucleic acids encoding the amino acid sequences depictedin Table 14a.

TABLE 14a Amino acid sequences of light chains (LC) and heavy chains(HC), with VH/VL domain exchange/replacement (1 + 1 CrossMAb^(Vh-V)) 1 +1 Antibody HC1 HC2 LC1 LC2 PD1TIM3_0389 SEQ ID SEQ ID SEQ ID NO: 52 SEQID NO: 50 NO: 51 NO: 53 PD1TIM3-0168 SEQ ID SEQ ID SEQ ID NO: 56 SEQ IDNO: 54 NO: 55 NO: 57 PD1TIM3-0476 SEQ ID SEQ ID SEQ ID NO: 64 SEQ ID NO:62 NO: 63 NO: 65 PD1TIM3-0477 SEQ ID SEQ ID SEQ ID NO: 68 SEQ ID NO: 66NO: 67 NO: 69 PD1TIM3_0166 SEQ ID SEQ ID SEQ ID NO: 60 SEQ ID NO: 58 NO:59 NO: 61

For all constructs knobs into holes heterodimerization technology wasused with a typical knob (T366W) substitution in the first CH3 domainand the corresponding hole substitutions (T366S, L368A and Y410V) in thesecond CH3 domain (as well as two additional introduced cysteineresidues S354C/Y349′C) (contained in the respective corresponding heavychain (HC) sequences depicted above).

Example 11B

Production and Expression of Multispecific Antibodies which Bind to PD1and TIM3 with VH/VL Domain Exchange/Replacement (2+2 CrossMAb^(Vh-VL))in Two Binding Arms and with Single Charged Amino Acid Substitutions inthe CH1/CL Interfaces

In an example multispecific antibodies which binds to human PD1 andhuman TIM3 were generated as described in the general methods section byclassical molecular biology techniques and were expressed transiently in293F of Expi293F cells as described above. The multispecific 2+2CrossMAb^(VH-VL) antibodies are described also in WO 2010/145792. Themultispecific antibodies were expressed using expression plasmidscontaining the nucleic acids encoding the amino acid sequences depictedin Table 14b.

TABLE 14b Amino acid sequences of light chains (LC) and heavy chains(HC), with VH/VL domain exchange/replacement (2 + 2 CrossMAb^(Vh-VL))2 + 2 Antibody HC LC1 LC2 PD1TIM3_0358 SEQ ID NO: 70 SEQ ID NO: 71 SEQID NO: 72 PD1TIM3_0359 SEQ ID NO: 73 SEQ ID NO: 74 SEQ ID NO: 75PD1TIM3_0321 SEQ ID NO: 76 SEQ ID NO: 77 SEQ ID NO: 80

Example 11C

Purification and Characterization of Multispecific Antibodies which Bindto PD1 and TIM3

The multispecific antibodies expressed above were purified from thesupernatant by a combination of Protein A affinity chromatography andsize exclusion chromatography. All multispecific antibodies can beproduced in good yields and are stable. The obtained products werecharacterized for identity by mass spectrometry and analyticalproperties such as purity by SDS-PAGE, monomer content and stability

Mass Spectrometry

The expected primary structures were analyzed by electrospray ionizationmass spectrometry (ESI-MS) of the deglycosylated intact CrossMabs anddeglycosylated/plasmin digested or alternatively deglycosylated/limitedLysC digested CrossMabs.

The VH/VL CrossMabs were deglycosylated with N-Glycosidase F in aphosphate or Tris buffer at 37° C. for up to 17 h at a proteinconcentration of 1 mg/ml. The plasmin or limited LysC (Roche) digestionswere performed with 100 μg deglycosylated VH/VL CrossMabs in a Trisbuffer pH 8 at room temperature for 120 hours and at 37° C. for 40 min,respectively. Prior to mass spectrometry the samples were desalted viaHPLC on a Sephadex G25 column (GE Healthcare). The total mass wasdetermined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik)equipped with a TriVersa NanoMate source (Advion).

Stability of Multispecific Antibodies

In order to assess stability of the antibody constructs, thermalstability as well as aggregation onset temperatures were assessedaccording to the following procedure. Samples of the indicatedantibodies were prepared at a concentration of 1 mg/mL in 20 mMHistidine/Histidine chloride, 140 mM NaCl, pH 6.0, transferred into a 10μL micro-cuvette array and static light scattering data as well asfluorescence data upon excitation with a 266 nm laser were recorded withan Optim1000 instrument (Avacta Inc.), while the samples were heated ata rate of 0.1° C./min from 25° C. to 90° C.

The aggregation onset temperature (T_(agg)) is defined as thetemperature at which the scattered light intensity starts to increase.The melting temperature (T_(m)) is defined as the inflection point in afluorescence intensity vs. wavelength graph. Results are shown in Table15.

TABLE 15 ProtA ProtA + prep SEC CE-SDS CE-SDS Yield main SEC Yield mainSEC Antibody [mg/L] peak monomer [mg/L] peak monomer T-agg MS PD1TIM3-21 88.9 97.6 19 92.1% 100% confirmed 0389 PD1TIM3- 48 87.3 88.9 42 98.4%95.2% confirmed 0168 PD1TIM3- 271 100 97.8 230 98.9% 100% confirmed 0476PD1TIM3- 211 94.3 85.3 159 97.4% 94.7% confirmed 0477

Example 12 Characterization of Anti-PD1-TIM3 Multispecific AntibodiesBinding Elisa ELISA for hu PD1

Nunc maxisorp streptavidin coated plates (MicroCoat #11974998001) werecoated with 25 μl/well biotinylated PD1-ECD-AviHis at a concentration of500 ng/ml and incubated at 4° C. over night. After washing (3×90 μl/wellwith PBST-buffer) 25 μl anti PD1 antibody samples were added inincreasing concentrations and incubated 1 h at RT. After washing (3×90μl/well with PBST-buffer) 25 μl/well goat-anti-human H+L-POD (JIR,JIR109-036-098) was added in 1:5000 dilution and incubated at RT for 1 hon a shaker. After washing (3×90 μl/well with PBST-buffer) 25 μl/well ofTMB substrate (Roche, 11835033001) was added and incubated until OD 2-3.Measurement took place at 370/492 nm.

ELISA for hu TIM3

Nunc maxisorp streptavidin coated plates (MicroCoat #11974998001) werecoated with 25 μl/well biotinylated TIM3-ECD-AviHis at a concentrationof 60 ng/ml and incubated at 4° C. overnight. After washing (3×90μl/well with PBST-buffer) 25 μl anti PD1 antibody samples were added inincreasing concentrations and incubated 1 h at RT. After washing (3×90μl/well with PBST-buffer) 25 μl/well goat-anti-human H+L-POD (JIR,JIR109-036-098) was added in 1:5000 dilution and incubated at RT for 1 hon a shaker. After washing (3×90 μl/well with PBST-buffer) 25 μl/well ofTMB substrate (Roche, 11835033001) was added and incubated until OD 2-3.Measurement took place at 370/492 nm.

ELISA results are listed as EC₅₀ values [nM] in Table 16.

TABLE 16 Biochemial-and Cell-binding of anti-PD1-TIM3 bispecificantibodies (ELISA) huPD1 huTIM3 Antibody Sample EC₅₀ [nM] EC₅₀ [nM] PD1IgG (bivalent) Chimeric PD1-0103 0.12 no binding TIM3 IgG ChimericTIM3-0018 no binding 0.15 (bivalent) 1 + 1 (bivalent) 1 + 1 PD1TIM3-01680.11 0.41 2 + 2 (tetravalent) 2 + 2 PD1TIM3-0359 0.09 0.11 TIM3 IgGChimeric TIM3-0028 no binding 0.29 (bivalent) upper plateau at 66% 1 + 1(bivalent) 1 + 1 PD1TIM3_0389 0.13 no binding 2 + 2 (tetravalent) 2 + 2PD1TIM3-0358 0.08 0.19 upper plateau at 65%

Avid binding (i.e. binding with both arms) can be detected forantibodies that are bivalent for Tim3 (Chimeric TIM3-0018, 2+2PD1TIM3-0359, Chimeric TIM3-0028, 2+2 PD1TIM3-0358). The higher EC50values for the 1+1 CrossMabs result from monovalent (towards Tim3),non-avid binding to the coated antigen. Avidity effects were notdetected for PD1-binding. EC50 values are comparable for bivalent andtetravalent formats.

Binding Biacore

Antigen Binding Properties of Multispecific Antibodies which Bind to PD1and TIM3

Binding of the multispecific antibodies to their respective targetantigens, i.e. PD1 and TIM3, was assessed by Biacore®.

PD1 Binding was Assessed According to the Following Procedure:

Anti-human Fc IgG was immobilized by amine coupling to the surface of a(Biacore) CM5 sensor chip. The samples were then captured and hu PD1-ECDwas bound to them. The sensor chip surface was regenerated after eachanalysis cycle. The equilibrium constant and kinetic rate constants werefinally gained by fitting the data to a 1:1 Langmuir interaction model.

About 10,000 response units (RU) of 20 μg/ml anti-human IgG (GEHealthcare #BR-1008-39) were coupled onto all flow cells of a CM5 sensorchip in a Biacore T200 using an amine coupling kit supplied by GEHealthcare. The sample and running buffer was HBS-EP+(0.01 M HEPES, 0.15M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20, pH 7.4). Flow celltemperature was set to 25° C. and sample compartment temperature to 12°C. The system was primed with running buffer.

Different samples were injected for 15 seconds with a concentration of10 nM and consecutively bound to the flow cells 2, 3 and 4. Then acomplete set of human PD1-ECD concentrations (300 nM, 100 nM, 2×33.3 nM,11.1 nM, 3.7 nM, 1.2 nM and 2×0 nM) was injected over each sample for300 s followed by a dissociation time of 10/600 s and two 30 sregeneration steps with 3 M MgCl₂, of which the last one contained an“extra wash after injection” with running buffer. Finally the doublereferenced data was fitted to a 1:1 Langmuir interaction model with theBiacore T200 Evaluation Software. Resulting K_(D), k_(a) and k_(d)values are shown in Table 17.

TIM3 Binding was Assessed According to the Following Procedure:

Anti-human Fab IgG was immobilized by amine coupling to the surface of a(Biacore) CM5 sensor chip. The samples were then captured and huTim3-ECD was bound to them. The sensor chip surface was regeneratedafter each analysis cycle. The equilibrium constant and kinetic rateconstants were finally gained by fitting the data to a 1:1 Langmuirinteraction model.

About 10,000 response units (RU) of 20 μg/ml anti-human Fab IgG (GEHealthcare #28-9583-25) were coupled onto all flow cells of a CM5 sensorchip in a Biacore T200 using an amine coupling kit supplied by GEHealthcare. The sample and running buffer was HBS-EP+(0.01 M HEPES, 0.15M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20, pH 7.4). Flow celltemperature was set to 25° C. and sample compartment temperature to 12°C. The system was primed with running buffer.

Different samples were injected for 30 seconds with a concentration of10 nM and bound consecutively to the flow cells 2, 3 and 4. Then acomplete set of human Tim3-ECD concentrations (600 nM, 200 nM, 2×66.7nM, 22.2 nM, 7.4 nM and 2×0 nM) was injected over each sample for 200 sfollowed by a dissociation time of 10/600 s and two 30 s regenerationsteps with Glycine HCl pH 2.1, of which the last one contained an “extrawash after injection” with running buffer. Finally the double referenceddata was fitted to a 1:1 langmuir interaction model with the BiacoreT200

Evaluation Software. Resulting K_(D), k_(a) and k_(d) values are shownin Table 17.

Results are indicated in Table 17.

TABLE 17 Affinity for PD1-Tim3 Bispecific Antibodies PD1-arm Tim3-armSample KD [nM] KD [nM] PD1TIM3-0389 (0357) 1.3 245 PD1TIM3-0358 (2 + 2)0.3 240 PD1TIM3-0168 1.2 10.3 PD1TIM3-0359 (2 + 2) 1.8 2.3 PD1TIM3-04760.1 332 PD1TIM3-0477 <0.1 12

All tested antibodies specifically bind to both targets, PD1 and TIM3,and exhibit an antigen affinity in the nanomolar range.

Example 13: FRET Assay for Simultaneous Binding of Anti-PD1/TIM3Bispecific Antibodies to Recombinant Cells

This example describes the development of a cell-based TR-FRET assay todetermine the simultaneous binding of bispecific antibody formats to twodifferent receptors present on one cell. The chosen Tag-lite technologyis a combination of a classical TR-FRET (time-resolved fluorescenceresonance energy transfer) and SNAP-tag technology (e.g. New EnglandBiolabs, CISBIO), which allows antigens present on the cell surface tobe labeled with a fluorescent donor or acceptor dye.

Aim of this Technology Evaluation

This assay is intended to demonstrate the simultaneous binding ofanti-PD1/Tim3 bispecific antibodies to cells expressing both PD1 andTim3 receptors as recombinant fusion proteins consisting of theextracellular domains (ECD) of the given receptor and a tag, to which afluorescence dye can bind. In the presence of a PD1-Tim3 bispecificantibody, which can bind both labeled receptors, the proteins will comeinto close proximity to allow energy transfer between the two FRET dyes(see FIG. 6).

Generation of recombinant PD1⁺TIM3⁺ HEK Cells

Standard methods were used to generation DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. For Cloning of PD1 andTIM3 variants SNAP or CLIP was inserted proximal to TM-region of thereceptor and cytoplasmatic domain was removed except for 7 aa andreplaced by Flag-tag.

Transient Transfection

HEK293 cells were co-transfected with transfection reagent 293free(Novagen) and Opti-MEM® I Reduced Serum Media (Life Technologies) in 30ml culture volume using two plasmids at a time with 15 μg total amountof DNA. Briefly, HEK293 cells were transiently transfected with thefollowing plasmids encoding for a fusion protein consisting of PD1 orTim3 ECDs and a SNAP or CLIP tag as described elsewhere:

Plasmid ID's and reference e.g. PD1-SNAP, Tim3-CLIP (the combinationPD1-CLIP and Tim3-SNAP was also constructed and expressed, but didresult in only low FRET signals).

Plasmids:

-   -   a) PD1-SNAP (in 5′ to 3′ direction):        -   nucleic acid encoding human PD1-extracellular domain            including the signal peptide (residue 1-170 of SEQ ID NO: 89            (30),        -   nucleic acid encoding GGGGS spacer (SEQ ID NO: 90),        -   nucleic acid encoding SNAP from pSNAP-tag(T7)2 (without            N-terminal methionine residue) which is a mutant form of the            human gene for O6-alkylguanine-DNA-alkyltransferase (hAGT).            (Compared to wild type hAGT, the SNAP-tag protein contains            the mutations C26A, K125A, A127T, R128A, G131K, G132T,            M134L, R135S, C150S, N157G, S159E, and is truncated after            G182) (SEQ ID NO: 91),        -   nucleic acid encoding GGGGS spacer (SEQ ID NO: 90),        -   nucleic acid encoding human PD1-transmembrane and            cytoplasmic domain (residue 171-191 of SEQ ID NO: 89),        -   nucleic acid encoding GGGGS spacer (SEQ ID NO: 90),        -   nucleic acid encoding the Flag-tag (DYKDDDDK; SEQ ID NO:            92).    -   b) Tim3-CLIP (in 5′ to 3′ direction):        -   nucleic acid encoding human Tim3-extracellular domain            including the signal peptide (residue 1-202 of SEQ ID NO:            93)        -   nucleic acid encoding GGGGS spacer (SEQ ID NO: 90),        -   nucleic acid encoding CLIP from pCLIPf (without N-terminal            methionine residue), which is a mutant form of the human            gene for 06-alkylguanine-DNA-alkyltransferase (hAGT) (SEQ ID            NO: 94),        -   nucleic acid encoding GGGGS spacer (SEQ ID NO: 90),        -   nucleic acid encoding human Tim3 transmembrane and            cytoplasmic domain (residue 203-230 of SEQ ID NO: 93)        -   nucleic acid encoding GGGGS spacer (SEQ ID NO: 90),        -   nucleic acid encoding the Flag-tag (DYKDDDDK; SEQ ID NO:            92).

Upon transfection, cells were incubated in shaker flasks until finalusage for FACS (24-48 hrs after transfection) or FRET experiments (after48 hrs).

Confirmation of PD1 and Tim3 Expression on Transiently TransfectedHEK293 Cells (FACS)

24-48 hrs after transient transfection of Hek293 cells, cells wereanalyzed for PD1 and Tim3 expression: Usually, 1-3×105 single or doubletransfected cells were stained 30 min on ice at 10 μg/ml, washed twotimes with PBS/2% FCS and analyzed on a FacsCanto II using

-   -   PD1-FITC Biolegend 329904, clone EH12.2H7) or PD1-PE (R&D        #FAB1086P) and/or    -   Tim3-PE (R&D FAB2365P clone 344823) and/or        Description Cell Labeling and FRET Assay with Anti-PD1/TIM3        Bispecific Antibodies

Description cell labeling and FRET assay with anti-PD1/TIM3 bispecificantibodies:

Transfected cells were sedimented and resuspended at a density of 1×106cells/ml in Tag-lite buffer (Cisbio). Then, cells were stained with 100nM SNAP-Lumi4-Tb (Cisbio) and 100 nM Clip-Red (Cisbio) for 1 h at 37° C.in Tag-Lite buffer (Cisbio). After washing and resuspension in PBS/2%FCS, about 50.000 cells (in 50p volume) were seeded into 96-wellflat-bottom white plates (Costar) bevor control (e.g. singlespecificity, isotype reference) or bispecific antibodies were added tothe cells at a final concentration of 0.001-10 nM. In some experiments,parental monoclonal antibodies were cross-linked via goat anti human Fc(20 nM final concentration, data not shown). After an incubation of 1 hat 4° C. or room temperature, time-resolved fluorescence was measured asratio of 665/620 nm with an BMG Pherastar reader or Tecan Infinite M1000Pro using standard settings provided by vendor. Optionally,SNAP-Lumi4-Tb and 100 nM Clip-Red labeled cells were stored at −80° C.or in liquid nitrogen and freshly thawed for FRET experiments.

Results: Characterization of Different Bispecific Antibodies andAntibody Formats for Simultaneous Receptor Binding and Cross-Linking asDemonstrated by FRET Induction.

PD1 and TIM3 expressing HEK cells were treated as described above tomeasure FRET signal upon simultaneous receptor binding via incubationwith titrated amounts of different bispecific antibodies (0.12-10 nM).

All bispecific antibodies induced a FRET signal in PD-1-TIM3-expressingcells in a dose-dependent manner. There was no dramatic differencebetween 1+1 formats (antibodies #389 and 168) compared to 2+2 constructs(358+359) as can be seen in FIG. 7A.

In addition, two bispecific formats based on humanised PD1 TIM3antibodies (#476 and 477) were also evaluated for their ability toinduce FRET in cells upon treatment. As demonstrated in FIG. 7B, bothconstructs induced significant FRET signal in PD1+ TIM3+ HEK cellsunderlining the simultaneous binding in a functional manner.

To Show the Specificity of the FRET Signal Induced by SimultaneousBinding of the Bispecific Antibody, Monoclonal IgGs of Only OneSpecificity were Added for Competition.

SNAP-tagged PD1 and CLIP-tagged TIM3 cells (as described before) werelabelled with 100 nM SNAP-Lumi4-Tb and 100 nM Clip-Red. After washing,labelled cells were incubated with the bispecific anti-PD1/TIM3 antibody#0168 [at indicated concentrations] for 1 h at 4° C. beforetime-resolved fluorescence was measured at 665/620 nm with an BMGPherastar reader (black lines). To underline the specificity of FRETsignal after bispecific antibody treatment, an anti-PD1 monoclonalantibody (#0165, FIG. 8A, grey curve) or an anti Tim-3 monoclonalantibody (#0018, FIG. 8B, grey curve) was added for competitionresulting in an almost complete prevention of the FRET signal. Theparental (monospecific) anti-PD-1 antibody alone did not induce FRET(dotted lines).

The induction of the FRET signal was also prevented in presence of aTIM3 parental antibody (0018; grey curves) added in parallel to thebispecific antibody (FIG. 8B).

Example 14: Binding of Antibodies to Different Peripheral BloodMononuclear Cells (PBMC)

Binding Assay

Freshly isolated PBMCs or 3 days polyclonally activated (plate boundanti-CD3 and soluble anti-CD28 antibodies, 1 ug/ml each, both from BDPharmingen) CD4 T cells were stained either with Alexa 647-directlyconjugated anti-TIM-3 or anti-TIM-3/anti-PD-1 bispecific antibodies for1 hour at 4 C degrees. The cells were then washed to eliminate unboundantibody and stained for surface markers for 30 minutes at 4 C degreesto discriminate monocytes (CD14⁺ (BD Pharmingen)), NK cells (CD16⁺(eBioscience), CD56⁺ (BioLegend) and CD3⁻) and T cells (CD3⁺(eBioscience)) before being fixed with BD Cell Fix. The cells wereacquired at LSRFortessa, BD Biosciences. Results for the bispecificantibodies in comparison to anti-Tim3 antibodies are shown in FIGS. 9Ato 9H.

Example 15: Internalization

Example 15A) Three days polyclonally activated CD4 T cells, previouslycultured with 1 mg/ml of plate bound anti-CD3 and 1 mg/ml of solubleanti-CD28 antibodies, were incubated in presence of either anti-TIM-3 oranti-TIM-3/anti-PD-1 bispecific antibodies (in duplicates) for 30minutes at 4° C. degrees. The cells were then washed, divided in twogroups, one of which incubated for 3 additional hours at 37° C. degreesand the other immediately stained with a labelled secondary antibody(eBioscience) before being fixed with BD Cell Fix. After the 3 hoursincubations also the second group of the cells were stained with thelabelled secondary antibody before fixation.

The cells were acquired at LSRFortessa (BD Biosciences) and theexpression levels of detectable antibody on the cell surface werecompared among the two groups. Results are shown in FIGS. 10A to 10D.Bispecific 1+1 PD1TIM3-0166 (based on chimeric PD1-0103/TIM3-0038)showed reduced internalization compared to bispecific 2+2 PD1TIM3-0321(also based on chimeric PD1-0103/TIM3-0038, but having two antigenbinding sites for PD and two for TIM3) and compared to parent TIM3-0038antibody on activated CD4+ T-Cells and on activated NK cells.

Example 15B) Visualization of Antibody Localization and Internalizationby Fluorescence Confocal Microscopy

Activated CD4-positive cells were stained with CMFDA (Molecular Probes,Life technologies), except when stained with a-PD1 antibody, and platedon round coverslips treated with Poly-L-Lysine (Sigma). Cells wereallowed 30 minutes to adhere at 37° C. before fluorescently-taggedantibodies (1 ug/mL: a-TIM3 (chi18-A647=cimeric Tim3_0018 labeled withAlexaA647), a-TIM3 (chi28-A647=cimeric Tim3_0028 labeled withAlexaA647), Bispec (0168-A647=1+1 PD1TIM3_0168 (based on chimericPD1-0103/Tim3-0018) labeled with AlexaA647) and Bispec (0389-A647=1+1PD1TIM3_0389 (based on chimeric PD1-0103/Tim3-0028) labeled with Alexa647) and a-PD1 (0165-A488=chimeric PD1-0103 labeled with Alexa488) wereadded directly into growth media for different durations (15 min, 1 hr,2 hr, 3 hr). Cold PBS (Lonza) was used to quench the reaction and towash off unbounded antibodies. Cells were then fixed (BD Cytofix) for 20minutes and washed twice with wash buffer (BD stain buffer). Aftertransferring the coverslips to a dry surface, they were then mounted onglass slides with mounting medium (Fluoromount G, eBioscience) and keptin the dark at 4° C. before imaging. The intensity of the fluorescentsignal from the membrane ROI, of highly targeted cells, was divided bythe intensity of the fluorescent signal from the cytoplasm ROI of thesame cells, resulting in a ratio displayed in the Box Charts. In orderto compare samples, One Way ANOVA analysis was used (*=p<0.05;**=p<0.001). Fluorescence confocal microscopy was performed with aninverted LSM 700 from Zeiss with a 60× oil objective. Images werecollected using Zen software (Zeiss) coupled to the microscope. Theanalysis of the images were performed with Imaris Software (Bitplane;Oxford Instrument) and the statistical analysis were performed byGraphPad Prism (Graphpad Software). The analysis over time showinghigher membrane localization in both bispecific and PD1 antibodies whencompared to intracellular clustering of TIM3 antibodies is shown inFIGS. 11A and 11B. The anti-PD1 and the Bispec 0389 show only very slowinternalization, even after 3 h, whereas the internalization for theother Bispec 0168 is stronger. Stronger internalization is shown byaTim3 Ab 0028, the most internalization is shown by aTim3-0018.

Example 16: T Cell Activation Via Mixed Lymphocyte Reaction (MLR) Assay

The Mixed Lymphocyte Reaction (MLR) is an immune cell assay whichmeasures the activation of lymphocytes from one individual (donor X) tolymphocytes from another individual (donor Y). A mixed lymphocytereaction was used to demonstrate the effect of blocking the PD1 pathwayto lymphocyte effector cells. T cells in the assay were tested foractivation and their IFN-gamma secretion in the presence or absence ofan anti-PD1/TIM3 bispecific mAbs.

To perform an allogeneic MLR, peripheral blood mononuclear cells (PBMCs)from at least four healthy donors of unknown HLA type were isolated bydensity gradient centrifugation using Leukosep (Greiner Bio One, 227288). Briefly, heparinized blood samples were diluted with the threefold volume of PBS and 25 ml aliquots of the diluted blood were layeredin 50 ml Leukosep tubes. After centrifugation at 800×g for 15 min atroom temperature (w/o break) the lymphocyte containing fractions wereharvested, washed in PBS and used directly in functional assay orresuspended in freezing medium (10% DMSO, 90% FCS) at 1.0E+07 cells/mland stored in liquid nitrogen. Individual 2-way MLR reactions were setup by mixing PBMCs from two different donors at a 1:1stimulator/responder cell ratio and co-cultures were done at least induplicate in flat-bottomed 96-well plates for 6 days at 37° C., 5% CO₂,in the presence or w/o of a different concentration range of purifiedbispecific PD1-TIM3 antibodies or their parental monospecific antibodies(either alone or in combination). Either no antibody or an isotypecontrol antibody was used as a negative control and rec hu IL-2 (20EU/ml) was used as positive control. After day 6 100 μl of medium wastaken from each culture for cytokine measurement. The levels ofIFN-gamma were measured using OptEIA ELISA kit (BD Biosciences).

The results are shown in Table 18A to 18D (IFN-γ secretion/release). Thebispecific PD1TIM3 antibodies promoted T cell activation and IFN-gammasecretion in concentration dependent manner. The value of % increase ofIFNγ secretion was calculated in relation to IFNγ production of MLR w/oadding of any blocking mAbs (basal allogeneic stimulation induced IFNγvalue as E−c) and MLR with adding of 20 EU/ml rec hu IL-2 (positivecontrol=100% IFNγ value as E+c) and was calculated according to formula:Rel. Stimulation [%]=((Example−E−c)/(E+c−E−c)*100.

Four separate experiments were performed:

In Experiment 1 the potency of PD1-TIM3 Bispecific Antibody 1+1PD1TIM3_0168 (based on chimeric PD1-0103/TIM3-0018 (=AB 0168) incomparison with chimeric PD1-0103 (=PD1-0165) and chimeric TIM3_0018(=Tim3-chi18) and combinations thereof was evaluated. Results are shownin FIG. 12A and Table 18A.

TABLE 18A EC₅₀ [nM] Antibody D2 + D6 aPD1-0165 (= chimeric PD1-0103) 7.7aTEV13-chi18 (= cimeric TIM3_0018) >274 Combo aPD1-0165 + aTIM3-chi181.7 Bispec AB 0168 (= 1 + 1 4.3 PD1TIM3 0168 (based on chimericPD1-0103/Tim3-0018)

In Experiment 2 the potency of PD1-TIM3 Bispecific Antibody 1+1PD1TIM3_0389 (based on chimeric PD1-0103/TIM3-0028 (=Bispec AB 0389) incomparison with chimeric PD1-0103 (=PD1-0165) and chimeric TIM3_0028(=TIM3-chi28) and combinations thereof was evaluated. Results are shownin FIG. 12B and Table 18B.

TABLE 18B EC₅₀ [nM] Antibody D2 + D6 aPD1-0165 (= chimeric PD1-0103) 6.5aTIM3-chi28 (= cimeric TIM3_0028) >274 Combo aPD1-0165 + aTIM3-chi18 1.5Bispec AB 0389 (= 1 + 1 PD1TIM3_0389 2.8 (based on chimericPD1-0103/TIM3- 0028)

In Experiment 3 the potency of PD1-TIM3 Bispecific Antibody 1+1PD1-0103/Ky8213 (based on chimeric PD1-0103/and anti-TIM3 Ky8213 from US2012/0189617 (see antibody8213 e.g. Example 33) which was producedanalogously as described in Example 1 as a 1+1 CrossMab) in comparisonwith chimeric PD1-0103 (=PD1-0165) and anti-TIM3-Ky8213 (from US2012/0189617 (see antibody 8213) e.g. Example 33) and combinationsthereof was evaluated. Results are shown in FIG. 12C and Table 18C.

TABLE 18C EC50 [nM] Antibody D2 + D6 aPD1-0165 (= chimeric PD1-0103) 6.0aTIM3-Ky8213 111 Combo aPD1-0165 + aTIM3-Ky8213 0.9 Bispec AB 1 + 1PD1-0103/TIM3- 4.6 Ky8213

In Experiment 4 the potency of PD1-TIM3 Bispecific Antibody 1+1PD1TIM3_0389 (based on chimeric PD1-0103/TIM3-0028 (=Bispec AB 0389(1+1))) in comparison with PD1-TIM3 Bispecific Antibody 2+2 PD1TIM3_0358based on chimeric PD1-0103/TIM3-0028 (=Bispec AB 0358 (2+2)), andchimeric PD1-0103 (=PD1-0165) and chimeric TIM3_0028 (=TIM3-chi28) andcombinations thereof was evaluated. Results are shown in FIG. 12D andTable 18D.

TABLE 18D EC₅₀ [nM] EC₅₀ [nM] Antibody D2 + D4 D1 + D3 aPD1-0165 (=chimeric PD1-0103) 5.7 5.7 aTIM3-chi28(= cimeric Tim3_0028) >264 >264Combo aPD1-0165 + aTim3-chi28 0.6 0.8 Bispec AB 0389 (1 + 1) (= 1 + 11.9 2.0 PD1TIM3_0389 (based on chimeric PD1-0103/Tim3-0028) Bispec AB0358 (2 + 2) (= 2 + 2 5.7 6.8 PD1TIM3_0358 (based on chimericPD1-0103/Tim3-0028)

TABLE 19 Summary of observed properties/results: Binding MLR CD4Internalization IFN-γ Antibody Monocytes NK cells T cells T cells CD4 Tcells ELISA Monospecific ++ +/− − ++ ++ +/− TIM3-0018 Bispecific 1 + 1++ + +/− +++ − +++ PD1TIM3_0168 Bispecific 2 + 2 ++++ +/− −/+ +++ −PD1TIM3_0359 Monospecific ++ +/− −/+ ++ + + TIM3-0038 Bispecific 1 + 1 ++/− +/− ++ − ++ PD1TIM3_0166 Bispecific 2 + 2 +++ +/− −/+ +++ ++ +++PD1TIM3_0321 Monospecific + +/− −/+ + ++ +/− TIM3-0028 Bispecific 1 + 1− − +/− +++ − +++ PD1TIM3_0389 Bispecific 2 + 2 ++ PD1TIM3_0358 Assay (− = no effect, +/− = very weak effect, + = weak effect, ++ = mediumeffect, +++ = strong effect, ++++ = very strong effect)

Exampe 17: Co-Culture of Antigen-Specific CD4 T Cells with BCell-Lymphoblastoid Cell Line ARH77

To investigate the effect of anti-PD-1 blockade on CD4 T cells inpresence of a MHCII-expressing tumor cell line we developed an assay inwhich freshly purified CD4 T cells are cocultured for 5 days in presenceof an EBV-immortalized B cell lymphoblast cell line (ARHI77). On the dayof the minimal mixed lymphocyte reaction (mMVLR), CD4 T cells wereenriched via a microbead kit (Miltenyi Biotec) from 10⁸ PBMCs obtainedfrom a healthy donor. Prior culture, CD4 T cells were labeled with 5 mMof carboxy-fluorescein-succinimidyl esther (CFSE). 10⁵ CD4 T cells werethen plated in a 96 well plate together with the B cell line (5:1) inpresence or absence of blocking anti-PD1 antibodies (either humanizedPD-1 0376, nivolumab or pembrolizumab), anti-TIM3 antibodies (eitherhumanized anti-TIM3 0438 or Kyowa-8213) or anti-PD-1/TIM3 bispecificantibody (humanized 0476) at the concentration of 10 μg/ml. Five dayslater we collected the cell-culture supernatants used to measure theIFN-γ levels by ELISA (R&D systems).

As shown in FIG. 13, we interestingly observed that anti-PD-1 treatmentsignificantly increased the ability of CD4 T cells to produce IFN-γ whencompared to untreated CD4 T cells (dashed line). In this assay theanti-PD-1 antibody 0376 has been equally able as the bench markantibodies in inducing the secretion of IFN-γ by CD4 T cells, whileanti-TIM3 antibody 0438 alone has only a marginal effect even ifstronger than the benchmark antibody Kyowa-8213.

Surprisingly, the bispecific antibody 0476 was better than thecombination of parental antibodies, anti-PD1 antibody 0376 alone and thebenchmark antibodies in driving IFN-γ secretion by CD4 T cells (P<0.01,one way ANOVA).

Example 18: Enhanced Efficacy of the PD1-TIM3 Bispecific Antibody InVivo

Immune suppressed female mice (NOG), aged 6-8 weeks at start of theexperiments, were challenged subcutaneously with 10⁶ MKN45 cells (humangastric carcinoma cell line, expressing high level of CEA) at day 0 inpresence of matrigel at 1:1 ratio. At day 7, PBMC from healthy humandonor were isolated: human heparinized blood was diluted ˜ 2:1 inphosphate buffer saline (PBS) (Gibco) and transferred into prepared 50ml Leucosep tubes, each containing 15 ml Histopaque-1077 (SigmaAldrich). After centrifugation (30 minutes, 450×g, RT, no brake), thePBMC bands were collected with a 5 ml pipette. Cells were transferredinto 50 ml tubes and washed with PBS (centrifugation at 350×g, 10 min).The washing step was repeated (centrifugation at 300×g, 10 min). Aftercentrifugation (10 min, 350×g), cells were re-suspended in RPMI medium.10⁷ PBMC were injected intravenously in the NOG mice creating amouse-human chimeric model. At day 10, a weekly scheduled therapy(vehicle or treatment with a compound selected from anti-PD1 (0376),Nivolumab, anti-TIM3 (0438) or anti PD1-TIM3 (0476)) started and wasgiven by intra-peritoneal injection. The treatment with the PD1-Tim3bispecific antibody (either 3 or 10 mg/kg; open triangle) was comparedwith equimolar (1.5 or 5 mg/Kg) concentration of the single agent PD1antibody (0376), of Nivolumab and of the Tim3 antibody (0438). Tumoursize was measured by Caliper in mm over a period of 30 days every 2-3days. In FIGS. 14A and 14B, the measurements of tumour volume are shownas mean volume within the group of mice.

All the treatments showed the capability to control tumor growth whencompared the vehicle treated group. The inhibition of only PD1 (by PD1antibody (0376) or the benchmark Nivolumab) and of only TIM3 antibody(0438), lead to a similar efficacy in controlling the tumor growth. Thisshows that by blocking either PD1 or TIM3 it is possible to enhance theanti-tumoral response. However, an increase of tumor growth inhibitioncan be observed when both PD1 and TIM3 are bound by the PD1-TIM3bispecific antibody. Whereas at low concentration a difference in tumorgrowth between bi-specific antibody and the other treatment cannot beobserved, at higher doses the inhibition of both PD1 and TIM3 by thetreatment with the PD1-TIM3 bispecific antibody results in a stronginhibition of tumour growth.

1. A bispecific antibody comprising a first antigen-binding site thatspecifically binds to PD1 and a second antigen-binding site thatspecifically binds to TIM3, wherein said first antigen-binding sitespecifically binding to PD1 comprises a VH domain comprising (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:38, and (iii) HVR-H3comprising an amino acid sequence of SEQ ID NO:39; and a VL domaincomprising (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:40; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:41,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42; andsaid second antigen-binding site specifically binding to TIM3 comprises(a) a VH domain comprising (i) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:1, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:2, and (iii) HVR-H3 comprising an amino acid sequence of SEQ ID NO:3;and a VL domain comprising (i) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:4 or SEQ ID NO:11 or SEQ ID NO:12, (ii) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:5, and (iii) HVR-L3 comprising theamino acid sequence of SEQ ID NO:6; or (b) a VH domain comprising (i)HVR-H1 comprising the amino acid sequence of SEQ ID NO:17, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:18, and (iii) HVR-H3comprising an amino acid sequence of SEQ ID NO:19; and a VL domaincomprising (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:20, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:21,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:22; or(c) a VH domain comprising (i) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid sequence of SEQID NO:30, and (iii) HVR-H3 comprising an amino acid sequence of SEQ IDNO:31; and a VL domain comprising (i) HVR-L1 comprising the amino acidsequence of SEQ ID NO:32, (ii) HVR-L2 comprising the amino acid sequenceof SEQ ID NO:33, and (iii) HVR-L3 comprising the amino acid sequence ofSEQ ID NO:34. 2.-22. (canceled)
 23. A method of producing the bispecificantibody according to claim 1, comprising the steps of a) transforming ahost cell with vectors comprising polynucleotides encoding saidbispecific antibody, b) culturing the host cell according underconditions suitable for the expression of the bispecific antibody and c)recovering the bispecific antibody from the culture. 24.-30. (canceled)31. The method of claim 23, wherein the bispecific antibody binds toTIM3 with an at least 50-fold lower binding affinity when compared tothe binding to PD1.
 32. The method of claim 23, wherein said firstantigen-binding site specifically binding to PD1 comprises (a) a VHdomain comprising the amino acid sequence of SEQ ID NO: 43 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 44, or (b) a VHdomain comprising the amino acid sequence of SEQ ID NO: 45 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 46, or (c) a VHdomain comprising the amino acid sequence of SEQ ID NO: 45 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 47, or (d) a VHdomain comprising the amino acid sequence of SEQ ID NO: 45 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 48, or (e) a VHdomain comprising the amino acid sequence of SEQ ID NO: 45 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 49, and thesecond antigen-binding site specifically binding to TIM3 comprises (a) aVH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 8, or (b) a VHdomain comprising the amino acid sequence of SEQ ID NO: 9 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 10, or (c) a VHdomain comprising the amino acid sequence of SEQ ID NO: 13 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 14, or (d) a VHdomain comprising the amino acid sequence of SEQ ID NO: 15 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 16, or (e) a VHdomain comprising the amino acid sequence of SEQ ID NO: 23 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 24, or (f) a VHdomain comprising the amino acid sequence of SEQ ID NO: 25 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 26, or (g) a VHdomain comprising the amino acid sequence of SEQ ID NO: 27 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 28, or (h) a VHdomain comprising the amino acid sequence of SEQ ID NO: 35 and a VLdomain comprising the amino acid sequence of SEQ ID NO:
 36. 33. Themethod according to claim 23, wherein the first antigen-binding sitespecifically binding to PD1 comprises a VH domain comprising the aminoacid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acidsequence of SEQ ID NO: 46, and the second antigen-binding sitespecifically binding to TIM3 comprises a VH domain comprising the aminoacid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acidsequence of SEQ ID NO: 16 or a VH domain comprising the amino acidsequence of SEQ ID NO: 25 and a VL domain comprising the amino acidsequence of SEQ ID NO:
 26. 34. The method according to claim 23, whereinthe first antigen-binding site specifically binding to PD1 comprises aVH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VLdomain comprising the amino acid sequence of SEQ ID NO: 46, and thesecond antigen-binding site specifically binding to TIM3 comprises a VHdomain comprising the amino acid sequence of SEQ ID NO: 25 and a VLdomain comprising the amino acid sequence of SEQ ID NO:
 26. 35. Themethod according to claim 23, wherein the bispecific antibody is ahuman, humanized or chimeric antibody.
 36. The method of claim 23,wherein the bispecific antibody comprises an Fc domain, a first Fabfragment comprising the antigen-binding site that specifically binds toPD1 and a second Fab fragment comprising the antigen-binding site thatspecifically binds to TIM3.
 37. The method of claim 36, wherein the Fcdomain is an IgG1 Fc domain or an IgG4 Fc domain.
 38. The method ofclaim 36, wherein the Fc domain comprises one or more amino acidsubstitution that reduces binding to an Fc receptor, in particulartowards Fc Q receptor.
 39. The method of claim 36, wherein the Fc domainis of human IgG1 subclass with the amino acid mutations L234A, L235A andP329G (numbering according to Kabat EU index).
 40. The method of claim36, wherein the Fc domain comprises a modification promoting theassociation of the first and second subunit of the Fc domain.
 41. Themethod of claim 36, wherein a first subunit of the Fc domain comprisesknobs and a second subunit of the Fc domain comprises holes according tothe knobs into holes method.
 42. The method of claim 36, wherein thefirst subunit of the Fc domain comprises the amino acid substitutionsS354C and T366W (EU numbering) and the second subunit of the Fc domaincomprises the amino acid substitutions Y349C, T366S and Y407V (numberingaccording to Kabat EU index).
 43. The method of claim 36, wherein in oneof the Fab fragments the variable domains VL and VH are replaced by eachother so that the VH domain is part of the light chain and the VL domainis part of the heavy chain.
 44. The method of claim 43, wherein in thefirst Fab fragment comprising the antigen-binding site that specificallybinds to PD1 the variable domains VL and VH are replaced by each other.45. The method of claim 36, wherein in one of the Fab fragments in theconstant domain CL the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat EU Index), and in the constant domain CH1 the aminoacids at positions 147 and 213 are substituted independently by glutamicacid (E) or aspartic acid (D) (numbering according to Kabat EU index).46. The method of claim 45, wherein in the second Fab fragmentcomprising the antigen-binding site that specifically binds to TIM3 theconstant domain CL the amino acid at position 124 is substitutedindependently by lysine (K), arginine (R) or histidine (H) (numberingaccording to Kabat EU Index), and in the constant domain CH1 the aminoacids at positions 147 and 213 are substituted independently by glutamicacid (E) or aspartic acid (D) (numbering according to Kabat EU index).47. The method of claim 23, wherein the bispecific antibody comprises(a) a first heavy chain comprising an amino acid sequence with at least95% sequence identity to the sequence of SEQ ID NO: 50, a first lightchain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 52, a second heavy chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 51, and a second light chain comprising anamino acid sequence with at least 95% sequence identity to the sequenceof SEQ ID NO:53, or (b) a first heavy chain comprising an amino acidsequence with at least 95% sequence identity to the sequence of SEQ IDNO: 54, a first light chain comprising an amino acid sequence with atleast 95% sequence identity to the sequence of SEQ ID NO: 56, a secondheavy chain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 55, and a second light chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO:57, or (c) a first heavy chain comprising anamino acid sequence with at least 95% sequence identity to the sequenceof SEQ ID NO: 58, a first light chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO: 60, asecond heavy chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO: 59, and a second lightchain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO:61, or (d) a first heavy chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 62, a first light chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 64, a second heavy chain comprising an amino acid sequence withat least 95% sequence identity to the sequence of SEQ ID NO: 63, and asecond light chain comprising an amino acid sequence with at least 95%sequence identity to the sequence of SEQ ID NO:65, or (e) a first heavychain comprising an amino acid sequence with at least 95% sequenceidentity to the sequence of SEQ ID NO: 66, a first light chaincomprising an amino acid sequence with at least 95% sequence identity tothe sequence of SEQ ID NO: 68, a second heavy chain comprising an aminoacid sequence with at least 95% sequence identity to the sequence of SEQID NO: 67, and a second light chain comprising an amino acid sequencewith at least 95% sequence identity to the sequence of SEQ ID NO:69. 48.The method of claim 23, wherein the bispecific antibody claim comprises(a) a first heavy chain comprising the amino acid sequence of SEQ ID NO:50, a first light chain comprising the amino acid sequence of SEQ ID NO:52, and a second heavy chain comprising the amino acid sequence of SEQID NO: 51, and a second light chain comprising the amino acid sequenceof SEQ ID NO:53, or (b) a first heavy chain comprising the amino acidsequence of SEQ ID NO: 54, a first light chain comprising the amino acidsequence of SEQ ID NO: 56, and a second heavy chain comprising the aminoacid sequence of SEQ ID NO: 55, and a second light chain comprising theamino acid sequence of SEQ ID NO:57, or (c) a first heavy chaincomprising the amino acid sequence of SEQ ID NO: 58, a first light chaincomprising the amino acid sequence of SEQ ID NO: 60, and a second heavychain comprising the amino acid sequence of SEQ ID NO: 59, and a secondlight chain comprising the amino acid sequence of SEQ ID NO:61, or (d) afirst heavy chain comprising the amino acid sequence of SEQ ID NO: 62, afirst light chain comprising the amino acid sequence of SEQ ID NO: 64,and a second heavy chain comprising the amino acid sequence of SEQ IDNO: 63, and a second light chain comprising the amino acid sequence ofSEQ ID NO:65, or (e) a first heavy chain comprising the amino acidsequence of SEQ ID NO: 66, a first light chain comprising the amino acidsequence of SEQ ID NO: 68, and a second heavy chain comprising the aminoacid sequence of SEQ ID NO: 67, and a second light chain comprising theamino acid sequence of SEQ ID NO:69.